EP0555405A4 - Specific detection of antibodies to human t-cell leukemia viruses - Google Patents

Abstract

Disclosed is a method and test kit for detecting in a biological sample an antibody to a virion of a human T-cell leukemia virus (HTLV) or to a cell infected with such a virion generally involving contacting the biological sample with an HTLV-II-encoded antigen and detecting formation of the antigen-antibody complex by an immunoassay. Also disclosed is a related method and test kit for detecting an HTLV-I virion or an HTLV-I-infected cell generally involving contacting the biological sample with the HTLV-I specific antigen, RP-B1, and detecting formation of the antigen-antibody complex by an immunoassay. The HTLV-II-encoded RP-IIB and RP-B2 peptides and purified nucleic acid encoding these peptides are also disclosed.

Description

SPECIFIC DETECTION OF ANTIBODIES TO HUMAN T-CELL LEUKEMIA VIRUSES

Background of the Invention This invention generally relates to peptides, methods, and kits for detecting antibodies to Human T- Cell Leukemia Viruses (HTLV's) or to HTLV-infected cells in a biological sample. In this application, we use the term "HTLV" to include two viruses, HTLV-I and HTLV-II, whose specific prototypes are disclosed in the articles cited below. We also include in that term variants, mutations, or other forms of HTLV-I and HTLV-II. We do not include the virus known as HIV (or HTLV-III) .

Human T-cell leukemia virus type I (HTLV-I) has been shown to be etiologically associated with human adult T-cell leukemia and lymphoma (ATLL) and with a neurological disorder termed HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) (see, e.g., B.J. Poiesz et al., Proc. Natl . Acad. Sci . USA 77..7415, 1980, S. Jacobson et al.. Nature 331:540, 1988). HTLV-I infection is endemic to Southwestern Japan and the Caribbean islands (P.H. Levine et al.. Int . J. Cancer 41: 1 , 1988). Estimates place the number of infected individuals in Japan at over one million (Y. Hinu a et al., AIDS Res . 2:517, 1986)

Human T-cell leukemia virus type II (HTLV-II) has been isolated, in two cases, from patients with an unusual T-cell malignancy resembling hairy cell leukemia (HCL) ; however, the etiological role of HTLV-II in this disease has not been established (see, e.g., I.S.Y. Chen et al.. Nature 305:502. 1983, J.D. Rosenblatt et al.. Leukemia .1:397, 1987). HTLV-II has also been isolated from individuals without evidence of malignancy, including a hemophiliac with an unexplained pancytopenia and a patient with acquired immunodeficiency syndrome

SUBSTITUTE SHEET (see, e.g., V.S. Kalyanaraman et al., EMBO J. 4.:1458, 1985)

The prevalence of both HTLV-I and HTLV-II infection appears to be increasing in the United States and Western Europe, particularly among intravenous drug abusers (H. Lee et al.. Science 244:471. 1989).

Shimotohno et al. (Proc. Natl . Acad. Sci . USA 3 '.3101, 1985) report the complete HTLV-II proviral DNA sequence and deduced amino acid sequence. Lee et al. (Proc. Natl . Acad. Sci . USA 81:7579, 1984) report that HTLV-II encodes an envelope glycoprotein, termed gp67.

Seiki et al. (Proc. Natl . Acad . Sci . USA 80:3618.

1983) report the HTLV-I proviral DNA sequence and deduced amino acid sequence.

Lee et al. (Proc. Natl . Acad. Sci . USA 81:3856,

1984) report that HTLV-I encodes a 61kD envelope glycoprotein (gp6l) .

Essex et al. (U. S. Pat. 4,743,678) discloses the use of the 61 kD HTLV-I envelope glycoprotein as a serologic marker for HTLV-I infection.

Schupbach et al. (Science 224:607. 1984) report that the HTLV-I envelope glycoprotein is cleaved into an amino-terminal 46kD glycoprotein (gp46) and a carboxy- terminal transmembrane 2ϊkD glycoprotein (gp21E) .

T. D. Copeland et al. (J. Immunol . 137:2945) report that a peptide derived from the carboxy-terminus of gp46 is recognized by HTLV-I-positive patient sera. Lee et al. (Proc. Natl . Acad. Sci . USA 81:3856, 1984) report that the HTLV-I envelope protein, which "appears to be the most immunogenic species in exposed people", is immunoprecipitated by serum samples from ATLL patients as well as from healthy carriers living in endemic areas. Lee et al. further report that human antisera known to have antibody reactivity to the gp61 envelope protein of

SUBST HTLV-l reacts equally well with the gp67 envelope protein of HTLV-II. Samuel et al. (Science 226:1094, 1984) report that a bacterially-expressed recombinant protein containing seven amino acids from the carboxy-terminal region of gp46 and two-thirds of the HTLV-I transmembrane protein is recognized by sera from HTLV-I-positive and HTLV-II-positive subjects.

There are various tests to determine the presence of antibodies to HTLV-I or HTLV-II in a biological specimen (Particle Agglutination Assay, Fujirebio Inc., Tokyo, Japan; EIA, Abbott Laboratories, North Chicago, IL; HTLV-I EIA, Dupont Company, Wilmington, DE) . In one particular example, an ELISA test is used for blood bank screening. For such assays, disrupted HTLV-I virions or recombinant HTLV-I virion proteins are used as the source of antigen for the ELISA test. This assay detects antibodies to HTLV-I and, at least, in some cases, also detects antibodies to HTLV-II. H. Lee et al. (Science 244A471, 1989) report that "serum was screened for antibodies to HTLV-I by enzyme immunoassay (EIA) (Abbott laboratories) . Although purified disrupted HTLV-I virions were used in the EIA, sera from two previously identified individuals infected with HTLV-II isolates (HTLV-II_M0 and HTLV-II_j^) were strongly cross-reactive in the HTLV-I EIA assay. Therefore, this assay would be expected to react with other HTLV-II antibody-positive sera". Anderson et al. (Blood 74:2585, 1989) report that "seroreactivity to both gp24 and gp46 and/or gp61 by WIB [Western I munoblot] or RIPA [radioimmunoprecipitation assays] or both are suitable criteria to confirm but not to distinguish HTLV-I and HTLV-II infections".

Summary of the Invention We have discovered that there may be a substantial population of HTLV-II-infected individuals, and we have

SUBSTITUTESHEET identified immunological markers to detect such individuals reliably, either as part of a general HTLV screen or as a specific assay to discriminate between HTLV-I and HTLV-II infection. By enabling reliable quantification of HTLV-II infection, our discovery identifies the scope of the problem being addressed and highlights the importance of the invention. The importance of our discovery is further highlighted by our finding that HTLV-I-encoded markers may fail to detect a non-trivial percentage of HTLV-II-positive samples.

In general, the invention features a method for detecting in a biological sample an antibody to a virion of a human T-cell leukemia virus (HTLV) or to a cell infected with the HTLV. The method involves providing an antigen encoded by HTLV-type II (HTLV-II) , contacting a biological sample with the antigen, and detecting formation of an antigen-antibody complex by immunoassay.

In a related aspect, the invention features a test kit for detecting in a biological sample an antibody to a virion of a human T-cell leukemia virus (HTLV) or to a cell infected with the HTLV. The kit is compartmentalized to receive in close confinement therein one or more containers which include: a first container containing an antigen encoded by HTLV type II (HTLV-II) , and a second container containing a means.for detecting the formation of an immunocomplex between the antibody and the antigen.

When the human T-cell leukemia virus detected is HTLV-II, the antigen is preferably all, or an antigenic segment or analog, of the HTLV-II envelope protein; preferably the carboxy-terminal half of the HTLV-II envelope protein; more preferably, RP-IIB or a segment or analog thereof which is reactive with RP-IIB antibodies; and, most preferably, RP-B2 or a segment or analog thereof which is reactive with RP-B2 antibodies. If

SUBSTITUTESHEE desired, however, it is possible to select and use HTLV- II-encoded antigens that are reactive with antibodies to HTLV-I.

In other preferred embodiments, the method further involves screening for both HTLV-I and HTLV-II. In addition to the use of the HTLV-II-encoded marker described above, such a screen involves contacting the biological sample with at least one antigen encoded by HTLV-type I (HTLV-I) , preferably an envelope segment or analog which is reactive with HTLV-I sera and not with HTLV-II sera, more preferably, RB-B1 or a segment or analog thereof which is reactive with RP-B1 antibodies. The antigen encoded by HTLV-II and the antigen encoded by HTLV-I may be combined and contacted with the biological sample simultaneously; or they may be maintained in separate containers and contacted with the sample sequentially or with different aliquots of the sample.

Detection of antigen-antibody complexes preferably is accomplished using a Western blot or an ELISA format. In another aspect, the invention features a method for discriminating between HTLV-I and HTLV-II infection. The method involves: providing HTLV-I recombinant protein RP-B1 or an antigenic segment or analog thereof, contacting the biological sample with RP-Bl or an antigenic segment or analog, and detecting formation of an antigen-antibody complex by immunoassay.

In a related aspect, the invention features a test kit for detecting in a biological sample an antibody to human T-cell leukemia virus type I (HTLV-I) . The kit is compartmentalized to receive in close confinement therein one or more containers which include: a first container containing HTLV-I recombinant protein RP-Bl or an antigenic segment or analog thereof, and a second container containing a means for detecting the formation of an immunocomplex between the antibody and the antigen.

sυ Detection of the immunocomplexes is preferably accomplished using a Western blot or an ELISA format.

The invention also features the RP-IIB peptide or a segment or analog thereof, preferably RP-B2, which is reactive with RP-IIB antibodies and, preferably, which is not reactive with antibodies specific for RP-Bl; and purified nucleic acid encoding the RB-IIB peptide or a segment or analog thereof which is reactive with RP-IIB antibodies. Preferably, such nucleic acid is included in plasmid, pIIB, or, more preferably, pB2.

By "antigen" is meant a substance, in this case, a peptide which interacts in a demonstrable selective way with an antibody (e.g., an antibody which is included in a biological sample) which results from challenge by all or part of the virus at issue. By "demonstrable selective way" is meant that the interaction of the antigen with the antibody is detectable (e.g., by an immunoassay) and the antigen is binds preferentially to the antibody directed against the virus at issue. A "peptide" is any chain of amino acids, including polypeptides and proteins as well as smaller peptide chains. By "antigenic segment" or "segment of an antigen" is meant any portion of an antigenic peptide which interacts in a demonstrable selective way with an antibody directed against the organism bearing the antigen. By "analog" is meant a peptide differing from the antigen or antigenic segment by one or more conservative or non-conservative amino acid changes; a suitable analog, as used herein, is one which interacts in a demonstrable selective way with an antibody directed against the organism bearing the antigen. Such analogs may be produced by any method of recombinant DNA technology or by chemical, e.g., peptide, synthesis. When we refer to "HTLV-I-encoded antigen" or "HTLV-II- encoded antigen", we include not only peptides whose

SUBSTITUTE SHEET amino acid sequence is encoded by a naturally occurring HTLV, but also analogs of such peptides as described above. By "purified " is meant substantially isolated from other cellular components, e.g., proteins, lipids, and other nucleic acids, with which the substance naturally occurs.

Other features and advantages of the invention will be apparent from the following detailed description, thereof, and from the claims. Detailed Description

The drawings are first briefly described. Drawings

FIG. 1 is the nucleic acid and corresponding amino acid sequence of the HTLV-II provirus (Shimotohno et al., Proc. Natl . Acad . Sci . USA 82:3101, 1985; SEQ ID NO.:l). FIG. 2 is a hydrophobicity plot of the HTLV-II- encoded envelope protein sequence and a comparison of the amino acid sequences of the HTLV-II and HTLV-I envelope proteins. FIG. 3 is the nucleic acid and corresponding amino acid sequence of HTLV-I including the sequence encoding RP-Bl (Seiki et al., Proc. Natl . Acad . Sci . USA 80:3618. 1983; SEQ ID NO. :3) .

FIG. 4 is a diagram depicting construction of expression plasmids, pB, pBl, and pD.

FIG. 5 is a diagram depicting construction of expression plasmids, pA and pC.

FIG. 6 is a diagram depicting construction of expression plasmid, pIIB. Obtaining HTLV-II-encoded antigens for use in the method of the invention

Those skilled in the art will recognize that HTLV- II-encoded serologic markers other than those disclosed below may be used to detect HTLV-II infection according to the invention. These serologic markers would preferably be envelope protein peptide segments or analogs, but may be any HTLV-II-encoded peptide which is reactive with antibodies to HTLV-II. To produce such peptide segments or analogs, HTLV-II-derived nucleic acid encoding putative antigenic peptides may be cloned into an expression vector (e.g., an expression vector described below) . Alternatively, such peptides may be synthesized by other techniques, including, without limitation, any technique of recombinant DNA technology or chemical, e.g., peptide, synthesis. One source of HTLV-II is the mammalian cell line, termed MO, which is permanently and persistently infected with HTLV-II; this cell line is available from the American Type Culture Collection (Accession No. CRL-8066) . Viral DNA may be isolated from such a cell line by standard techniques

(see, e.g., Tsujimoto et al., Λfol. Biol . Med. 5.:29, 1988; Seiki et al., Proc. Natl . Acad. Sci . USA 79:6899. 1982; Hirt, B., J. Mol . Biol . 2j5:365, 1967). Alternatively, HTLV-II DNA may be obtained from available full-length or partial HTLV-II proviral clones (e.g., AH6.0; Chen et al.. Nature 305:502, 1983; Shimotohno et al. , Proc. Natl . Acad. Sci . USA 82:3101, 1985). The full-length HTLV-II sequence is shown in Fig. 1 (SEQ ID N0.:1)

Information regarding hydrophobicity provides guidance in choosing candidate antigenic peptides (including antigenic peptide segments or analogs) ; hydrophilic regions are more likely to be antigenic, i.e., reactive with HTLV-II-positive sera, because these regions are exposed on the surface of the virion or virion-infected cell. A hydrophobicity plot of the HTLV- II envelope protein sequences is shown in Fig. 2. To facilitate selection of candidate peptides (including peptide segments or analogs) which specifically or selectively react with HTLV-II-or HTLV-I-positive sera, Fig. 2 also shows the the regions of conserved amino acid

SU residues along the HTLV-I and HTLV-II amino acid sequences. Conserved segments of amino acids (i.e., identical amino acids) are represented by black boxes. Peptide segments derived from regions of divergent sequence would be most likely to react specifically with serum directed to one particular type of HTLV (i.e., either HTLV-I or HTLV-II) .

Candidate peptides may be tested, by methods described in greater detail below, using, e.g., PCR- confirmed HTLV-II-positive antiserum (as described below) . Antigenic peptides are those which react with HTLV-II-positive serum (as determined using the methods described below) . Peptides specific for HTLV-II are those which react exclusively or selectively (e.g., a "two plus antibody reactivity", as defined below) with HTLV-II-positive serum.

A particularly preferred HTLV-II-encoded antigen is the recombinant protein, RP-IIB (described below; SEQ. ID NO. : 2) or segments or analogs thereof. Candidate segments or analogs may be produced, e.g., by any technique of recombinant DNA technology or chemical synthesis and screened for antigenicity, i.e., reactivity with HTLV-II-positive serum, using methods well known to those skilled in the art, e.g., those methods described below. One preferred antigenic segment of RP-IIB is included in the peptide RP-B2 (described below) . HTLV-I-encoded antigens

Those skilled in the art will recognize that many HTLV-I-encoded antigens (either purified or provided in mixtures obtained from disrupted virions or virion- infected cells) can be used to detect HTLV infection. Where the goal is to screen samples for infection by either HTLV-I or HTLV-II, the HTLV-I-encoded antigen can be used in addition to the above-described HTLV-II- encoded antigen to reduce the incidence of false negative

SUBSTITUTESHEET readings. In that case, it is possible to combine any of a number of known human HTLV-I serologic markers with an HTLV-II-encoded antigen, e.g., as discussed above.

Where the goal is to distinguish HTLV-I from HTLV- II, it is desirable to use RP-Bl, as described below, or a segment or analog thereof. Candidate segments or analogs of RP-Bl would be generated and screened for antigenicity as described above for RP-IIB, and for lack of reactivity with HTLV-II. Sources of HTLV-I include the mammalian cell lines, termed MJ and C5-MJ, which are permanently and persistently infected with HTLV-I; these cell lines are available from the American Type Culture Collection (ATCC Accession Nos. CRL-8294 and CRL-8293, respectively) . Viral DNA may be isolated from such cell lines by standard techniques (see, e.g., Tsuji oto et al., Mol . Biol . Med. .5:29, 1988; Seiki et al., Proc . Natl . Acad. Sci . USA 19.:6899, 1982; Hirt, B., J. Mol . Biol . 2 :365, 1967). Alternatively, HTLV-I DNA may be obtained from available full-length or partial HTLV-I proviral clones (e.g., λATM-1 or λATK-1; Seiki et al., Proc. Natl . Acad. Sci . USA 80.3618, 1983) . The HTLV-I sequence encoding RP-Bl is shown in Fig. 3 (SEQ ID NO.:3) Experimental Information

The following specific examples are provided to illustrate, not to limit, the invention. .

The preferred HTLV serologic markers according to the invention are identified from a panel of recombinant proteins containing different regions of the HTLV-I or HTLV-II env proteins whose immunological reactivity is assayed as described below.

Serologic Reactivity to HTLV-I Recombinant Proteins

To produce a test panel of recombinant proteins, fragments of HTLV-I proviral DNA sequences were cloned into appropriate expression vectors, using as a reference, the HTLV-I sequence reported by Seiki et al. (Proc. Natl . Acad. Sci . USA 8J):3618, 1983, hereby incorporated by reference) . The intact env gene was carried on the plasmid, psphl-envl. This plasmid was constructed as follows. The plasmid pMT2 (Clark et al., Nature 305:60. 1983, hereby incorporated by reference) was digested with SphI and a 4.2-kilobase (kb) fragment containing both the HTLV-1 env gene and the x region was isolated and subcloned into expression vector, p806 (a derivative of pUClδ containing the tac promoter and v- rast gene on an EcoRI-NcoI fragment which was originally derived from plasmid pXVR; Feig et al., Proc . Natl . Acad. Sci USA £33.:4607, 1987, hereby incorporated by reference). The inserted env gene was determined by sequence analysis (by the procedure of Toneguzzo et al., Biotechniques (5:460, 1988, hereby incorporated by reference) to be in the same reading frame as the preceding v-rasⁿ gene.

The distal two-thirds of the HTLV-I env gene was expressed from plasmid, pS3. To generate pS3, psphl- envl was digested with Sail, and a SalI-ended fragment containing the first 5694 nucleotides of the env gene was deleted. The resultant plasmid (containing nucleotides 5694 to 6665 of the env gene) was treated with DNA polymerase Klenow fragment and religated by blunt-end ligation (Fig. 4) . The transmembrane env protein (encoded by nucleotides 6140 to 6665) , was expressed from plasmid pD (Fig. 4) . To generate pD, psphl-envl DNA was digested with Kpnl , and a JCpnl-ended fragment containing the first 6140 nucleotides of the env gene was deleted. The plasmid DNA (containing nucleotides 6140 to 6665 of the env gene) was treated with DNA polymerase Klenow fragment and religated by blunt end ligation.

Two internal regions of the HTLV-I env gene were expressed from plasmids, pB and pBl (Fig. 4) . These plasmids were generated from plasmid pS3, isolated from

T E. coli strain JM110 and digested with either Clal or Xhol . Ends were blunted with DNA polymerase Klenow fragment and re-ligated, by blunt-end ligation, in the presence of Nhel nonsense codon linker DNA, d(CTAGCTAGCTAG) (New England Biolabs, Beverly, Mass.) The resultant plasmids, pB and pBl, contained HTLV-1 env gene nucleotide sequences (5694 to 5887) and (5694 to 5799; SEQ ID NO.: 3), respectively (Fig. 4).

Expression vector pJL6 (Lautenberger et al.. Gene Anal . Technol . 41: 49 , 1984, hereby incorporated by reference) was used to express the N-terminal half of the HTLV-I envelope glycoprotein, gp46. pJL6 contains a bacteriophage λ p_L promoter and the N-terminal fragment of the λ ell gene, including the ribosome-binding site and an ATG start codon. As shown in Fig. 5, a 422-base- pair (bp) Pvull-Sall fragment (including nucleotides 5273 to 5694) was obtained from pMT2 (Clark et al.. Nature 305:60, 1983). It was then treated with DNA polymerase Klenow fragment and fused in-frame to plasmid vector pJL6 (Lautenberger et al., Gene Anal . Technol . .42:49, 1984), which had been digested with Jϊindlll and blunt-ended using DNA polymerase Klenow fragment. The resultant plasmid was termed plasmid pA (Fig. 5) . Plasmid pC (Fig. 5) , encoding the C-terminal region of gp46, was constructed by inserting a 234-bp Clal-BamHI fragment (including nucleotides 5887 to 6120) from plasmid pMT2 (Clark et al., Nature 305:60. 1983) into plasmid vector pJL6, which had been previously linearized by digestion with Clal and BamEI (Fig. 5) . The recombinant protein, RP-IIB, was expressed from plasmid, pIIB. This plasmid was constructed by isolating, from plasmid pMOlA (Gelmann et al. , Proc. Natl . Acad. Sci . USA £1:993, 1984, hereby incorporated by reference), a 0.42 kb __sal-_.sal fragment containing an internal region (i.e., nucleotides 5462-5883; SEQ ID NO.:

SUBSTITUTESHEET 1) of the HTLV-II env gene (nucleotides 5180-6130; SEQ ID NO. : 1) and subcloning this fragment into plasmid pJL6 (Lautenberger et al.. Gene Anal . Technol . 42.:49, 1984), which had been digested with Clal and blunt-ended using DNA polymerase Klenow fragment (Fig. 6) . The inserted env gene was shown to be in the same reading frame as the preceding lambda phage ell gene by DNA sequence analysis using the procedures of Toneguzzo et al. (Biotechniques .6:460, 1988). To clone the RP-IIB-encoding fragment into" another vector (e.g. , other prokaryotic expression vectors or a eukaryotic expression vector) , pMOlA may be digested, as described above, with Rsal and a 420 base pair fragment isolated for insertion into the desired vector. Recombinant proteins (RPs) containing specific regions of HTLV-1 gp61 were produced by either vector p806 (pB, pBl, and pD) or by vector JL6 (pA and pC) in two different bacterial culture systems. E. coli X-90 (Pallas et al., J. Virol . 40:1075, 1986, hereby incorporated by reference) was transformed with p806- derived plasmids and the RPs were produced following induction with isopropyl-ø-D-thiogalactopyranoside (IPTG) as described in Matsuda et al. (Proc. Natl . Acad. Sci . USA .85:6968, 1988, hereby incorporated by reference). E. coli DC1148 (Lautenberger et al.. Gene Anal . Technol .

41: 49 , 1984), a strain lysogenic for a lambda phage which encodes the mutant temperature-sensitive repressor, cI857, was transformed with pJL6-derived plasmids and the RPs were produced following induction upon temperature shift from 32° to 42°C as described in Samuel et al. (Science 226:1094. 1984, hereby incorporated by reference) .

Following induction of recombinant protein production, the RPs were partially purified as described in Matsuda et al. (Proc. Natl . Acad . Sci . USA 85:6968,

SUBSTITUTESHEET 1988) and were identified by Coomassie blue staining (as described in Reisner et al., Anal . Biochem . 64.:509, 1975, hereby incorporated by reference) of cellular proteins which had been separated by electrophoresis on a sodium dodecyl sulfate-polyacrylamide (SDS-PA) gel (Laemmli, Nature 227:680_f 1979). Their molecular weight (in kDa) was determined by comparison with protein standards (of known molecular weight) ; the apparent molecular weight of each RP is given in Table 1.

TABLE 1

10 N, N terminus; M, middle region; and C, C terminus of gp.6, the exterior domain of gp61; gp21, the transmembrane domain of gp61. In most instances, the observed mass deviated slightly from the predicted mass. The predicted mass was calculated from the DNA sequence. ^c Values are rough estimates of the relative quantities of recombinant proteins expressed against a background of total bacterial proteins as observed in Coomassie blue-stained SDS-PA gels.

I

Plasmids psphl-envl and pS3 failed to express their RPs. However, the truncated protein fragments were expressed at levels high enough to allow direct detection by Coomassie blue staining. The RPs ranged from 15 to 32 kDa and together covered approximately 98% of the total HTLV-1 env precursor protein. The pJL6 bacterial system generally produced the RPs in greater quantity than did the p806 system, however, the reason for this difference is unclear. Identification of the RPs was confirmed by Western blot (WB) analysis using high-titer HTLV-l-positive sera and goat anti-v-ras^H antiserum which recognized the fusion proteins expressed by pB, pBl, and pD. By this assay, the observed molecular masses of the RPs also corresponded to their predicted molecular weight values. RP-D consisted of two molecular weight species, one of molecular weight 32 kDa, the other of 30 kDa; both reacted on Western blots with most HTLV-l-positive sera and with a rabbit anti-gp21 antiserum. RP-IIB was produced by transformation of pIIB into E. coli strain DC1148. An 18 kD protein was expressed by pIIB and was shown to be reactive to an HTLV-II-positive serum by WB assay 20 minutes after the temperature shift. Its production reached a plateau 40 minutes after temperature shift. To purify the protein, the bacterial pellet was subjected to a series of partial purification steps which included successive high salt and detergent extractions combined with sonication as described in Matsuda et al. (Proc. Natl . Acad . Sci . USA 85:6968, 1988) . RP-IIB was subsequently detected in an 8M urea fraction by Coomassie blue staining of an SDS- polyacrylamide gel (Laemmli, Nature 227:680. 1979, hereby incorporated by reference) .

Serum samples from two groups of Japanese HTLV-1- positive subjects, healthy carriers, and ATL patients

SUBSTITUTESHEET were used to study the prevalence of antibodies to each of the specific RPs. Initial screening for HTLV-1 seropositivity was standardized by using both the particle agglutination test (Ikeda et al., Gann . 75:845, 1984, hereby incorporated by reference) and the immunofluorescence test (Ishizaki et al., J. AIDS 1:340, 1988, hereby incorporated by reference) . All samples were then confirmed as positive by both a Western blot assay (as described in Barin et al.. Lancet ii:1387. 1985, hereby incorporated by reference) using MJ cell lysates (Popovic et al.. Science 219:856. 1983, hereby incorporated by reference) and a radioimmunoprecipitation assay (RIP assay) using [³⁵S]cysteine-labeled MT-2 antigens (Miyoshi et al., Nature 294 : 110. 1981, hereby incorporated by reference) .

As shown in Table 2, antibody reactivity to the RPs can be summarized as follows: 22.2% (10 of 45) of samples from the carriers and 16.9% (13 of 77) of samples from the ATL patients were reactive to RP-A; 80% (36 of 45) of samples from the carriers and 68.8% (53 of 77) of the samples from the ATL patients were reactive to RP- Bl; and 91.1% (41 of 45) of samples from the carriers and 93.8% (76 of 81) of samples from the ATL patients were reactive to RP-C.

SUBSTITUTE SHEE TABLE 2

Reactivity ^a [no. positive/no. tested (X positive)] with:

Serum source

RP-A RP-B RP-B1 RP-C RP-D RP-B + RP-C RP-A + RP-B + RP-C

HTLV-1 carriers 10/45 (22.2) 36/45 (80.0) 32/44 (72.7) 41/45 (91.1) 25/45 (55.6) 43/45 (95.6) 44/45 (97.8)

ATL patients 13/77 (16.9) 76/80 (95.0) 53/77 (68.8) 76/81 (93.8) 59/69 (85.5) 80/81 (98.8) 81/81 (100) 10 Total 23/122 (18.9) 112/125 (89.6) 85/121 (70.2) 117/126 (92.9) 84/114 (73.7) 123/126 (97.6) 125/126(99.2)

. Rates of reactivity to RP-D versus RP-B plus RP-C were statistically significant (P<0.005).

The antibody reactivity rates for all the serum samples, as assayed by Western blot, were as follows: 18.9% (23 of 122) reacted with RP-A; 89.6% (112 of 125) reacted with RP-B; 70.2% (85 of 121) reacted with RP-Bl, and 92.9% (117 of 126) reacted with RP-C. These results indicated that the C-terminal half of gp46 (RP-B plus RP- C) detected 97.6% (123 of 126) of the HTLV-II-positive sera, a percentage which is significantly higher (P<0.005) than that detected by the N-terminal half of gp46 (i.e., RP-A).. RP-A, RP-B, and RP-C, which together span the entire length of gp46 except the first five amino acids at the N-terminus and the last four amino acids at the C-terminus, detected 99.2% (125 of 126) of the HTLV-I-positive subjects. This same combination of RPs detected every ATL patient in this study. In contrast, RP-D, which contains the transmembrane envelope protein gp21 minus the first amino acid at the N- terminus, had only a 73.7% (84 of 114) reactivity rate. The difference in the rates of antibody response between gp46 (RP-A plus RP-B plus RP-C) and gp21 (RP-D) is statistically significant (P<0.005; Statistical Methods in the Biological and Health Sciences, ed. Milton and Tsokos, McGraw-Hill Book Co., 1983). Recombinant Protein B2 contains an HTLV-II-specific Antigen

To produce expression plasmid, pB2, a DNA fragment including nucleic acid residues 5675 to 5801 was ligated, by blunt end ligation, into Smal-/ Sphl-digested p806; both the RP-B2-encoding fragment and the p806 vector were treated with Klenow fragment to produce blunt ends prior to ligation. The 0.16 kb RP-B2-encoding fragment was produced by standard methods of PCR amplification, using plasmid pIIB as a template and the primers: 5' GCGGAATTCGTATGATCCTTTATGGTTC 3' (SEQ ID NO.: 4) AND

SUBSTITUTE SHEET 5' GCGGCATGCCTAGGTCAGCTGGATAAATTT 3¹ (SEQ ID NO.: 5) Plasmid pB2 encodes a fusion protein, termed RP-B2, containing 48 amino acids from the HTLV-II envelope protein (i.e., amino acid residues 166 to 213 of Fig. 1; SEQ ID NO.: 1) .

RP-B2 showed specificity for HTLV-II-positive sera in WB assays. None of the 12 HTLV-I-positive sera, shown above to cross-react with RP-IIB, had detectable antibody reactivity to RP-B2; all 20 HTLV-II-positive sera exhibited antibody reactivity to RP-B2. Recombinant Protein Bl contains an HTLV-I-specific Antigen

Twenty PCR-confirmed HTLV-II-positive serum samples (from intravenous drug abusers from New Orleans; Serologicals, Inc., Marietta, GA) were used to study antibody reactivity to five of the recombinant proteins (RPs-A, -B, -Bl, -C and -D) , described above. PCR confirmation was carried out by analyzing isolated lymphocyte DNA according to the ³²[P]-oligonucleotide end- labelling PCR technique described previously (Lee et al.. Science 244:471.1989, hereby incorporated by reference) Five oligonucleotide primer pairs corresponding to tax, protease and LTR sequences were utilized. The tax oligonucleotide primers corresponded to conserved sequences in both HTLV-I and HTLV-II; the two protease primer pairs and the two LTR primer pairs distinguish absolutely between HTLV-I and HTLV-II (Duggan et al.. Blood 21:1027, 1988). RPs were analyzed for antibody reactivity by Western blotting (WB) as described above. Partially purified RPs solubilized either in 3M or 8M urea were subjected to electrophoresis on a 15% SDS- polyacrylamide gel (Laemmli, Nature 227:680. 1970 and passively transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, NH) for 36 hours using the

SUBSTITUTESHEET methods of Barin et al. (Lancet ii:1387, 1985). WB strips were then lined up and the reactive bands were scanned using a video-densitometer scanner (Model 620, BioRad Laboratories, Richmond, CA) . When appropriate, the radioimmunoprecipitation (RIP) procedure of Chen et al., (J. Virol . 63.:4952, 1989, hereby incorporated by reference) was used as a confirmatory test; this test utilizes MT-2, an HTLV-I cell line (Miyoshi et al., Nature 294 : 110. 1981. The HTLV-I specific viral antigens expressed in MT-2 cells have been reported previously. Lee et al., In Human T-cell Leukemia /Lymphoma Virus , ed. Gallo et al., pp. 111-120, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1984) .

Antibody reactivity with each of the RPs were determined to be as follows: 10% (2/20) for RP-A; 5%

(1/20) for RP-B; 0% for RP-Bl; 85% (17/20) for RP-C; and 80% (16/20) for RP-D. RP-IIB, which contained amino acid residues 96-235 from the HTLV-II exterior envelope glycoprotein (i.e., amino acid residues 21-308) was reactive to all 20 HTLV-II serum samples.

To more carefully compare antibody reactivity, RP- Bl and RP-IIB proteins were used in a Western blot assay to study the antibody reactivity of 115 additional plasma samples. Those samples included: 9 PCR-confir ed HTLV- I and 45 PCR-confirmed HTLV-II plasma samples from intravenous drug abusers in New Orleans, 13 HTLV-I-Enzyme immunoassay negative samples, l HTLV-I-WB indeterminate case, and 1 HTLV-I seropositive but PCR-negative case. Another panel of 46 PCR-confirmed HTLV-I plasma samples were selected from a nationwide survey of food service employees in 1987-1988 in Jamaica. The samples were randomly mixed, blind-coded by a collaborator from another laboratory, and tested by WB assay. The results of the WB test were classified into two major categories: seropositive to RP-Bl (presumably, HTLV-I-positive cases)

SUBSTITUTESHEET or seronegative to RP-Bl (presumably, HTLV-I-negative cases) . In each major category, samples were divided into four subcategories (negative, one plus, two pluses and three pluses) , according to their antibody reactivities to RP-IIB. These relative levels of antibody reactivity were determined by densitometer scanning. Following such categorization, the serum sample code was broken, and the results of the WB tests were shown to be identical to the PCR results.

TABLE 3

Geographic Origin*** RP-WB* HTLV-I* New Orleans

c D B1-

10 6 14 25

Total 55 45

15 O * Western blot assays done under blind code using recombinant protein (RP) B1 or MB which contains 36 or 140 amino acids from middle regions of HTLV-I Z or HTLV-II gp46, respectively. 1 ** Determined by PCR with 5 oligonucleotide primer pairs corresponding to tax, protease and LTR regions. *** Food handlers from Jamaica; IVDAs from New Orleans.

As shown in Table 3, all 55 PCR-confirmed HTLV-I- positive samples and none of the 45 PCR-confirmed HTLV- II-positive samples were reactive to RP-Bl. All 45 HTLV- II samples were reactive with RP-IIB, and most of the positively scoring antibody reactivity was scored as two to three pluses (39/45) . Thirty-six of 55 (65%) HTLV-I- positive samples were cross-reactive to RP-IIB, however, the majority of these samples exhibited a one-plus antibody reactivity. Among the 13 HTLV-seronegative samples, there were 3 cases (21%) which had antibody reactivities to RP-IIB; none had antibody reactivity to RP-Bl. These 3 cases were found to have indeterminate antibody profiles to HTLV-I antigens by radioimmunoprecipitation assay (using MT-2 cells and the procedure described in Lee et al. , Proc . Natl . Acad . Sci . USA 81:7579, 1984). One case, which had two-plus antibody reactivity to RP-IIB, had antibodies to HTLV-I gag pl9. The other two cases which had two-plus and one- plus antibody reactivities to RP-IIB, respectively, had antibodies to HTLV-I gag p55. These three cases may represent HTLV-II infections which have been neglected by the current HTLV-I antigen-based serological tests. In addition, a WB-indeterminate case and an HTLV-I- seropositive but HTLV-I and HTLV-II PCR-negative case also show antibody reactivity to RP-IIB. .

Recombinant protein IIB contains HTLV-II-specific epitopes

Serum samples from the following groups were tested for their antibody reactivities to RP-IIB: 27 HTLV-II carriers from New Orleans; a patient MO from whom the first HTLV-II was isolated (Chen et al.. Nature 305:502, 1983); 20 HTLV-I carriers from an endemic area in Japan; 17 ATL patients from an HTLV-I endemic area in Japan (Chen et al., J. Virol . 62:4952, 1989);. and 10 HTLV-I/II negative blood donors. All serum samples were tested by two HTLV-I enzyme immuno assays (Abbott EIA, Abbott Laboratories, North Chicago, IL and Du Pont EIA (Du Pont Company, Wilmington, DE) , and confirmed by WB and radioimmunoprecipitation (RIP) (using the procedures cited above) . HTLV-II carriers were confirmed by PCR according to the technique of Lee et al. (Proc. Natl . Acad. Sci . USA 31 : 1519 , 1984) described above. Among 27 HTLV-II carriers confirmed by PCR, two carriers were seronegative in Abbott and Du Pont HTLV-I EIAs and had indeterminate antibody profile for HTLV-I infection

(i.e., one case only had anti-Gag p24 antibody; another case only had anti-Env gp61 antibody) .

TUTE SHEET TABLE 4

Reactivity (number positive) Rates of

5 Serum source (number) +++ Seropositivity to RP-IIB

100% 50% 24%

a. The intensity of reactivity to RP-IIB was classified into +, ++ and +++ according to the O.D. (absorption reading by densitometer scanning) of each sample in Western blot test. ω rn _ q

As shown in Table 4, in addition to patient MO, all 27 PCR-confirmed HTLV-II carriers had antibody reactivity to RP-IIB. In contrast, only 10 of 20 HTLV-I carriers and 4 of 17 ATL patients had antibody reactivities to RP-IIB. None of the 10 normal human sera had antibody reactivity to RP-IIB. The differences in the rates of seropositivity to RP-IIB between HTLV-II infected people (100%) and HTLV-I carriers (50%) , or ATL patients (24%), or all HTLV-I infected persons (14/37, 37.8%) were highly statistically significant (by Fisher's exact test, two-tail; p=3.57E-05, p=4.58E-08 and p=4.30E- 08, respectively; Statistical Methods in the Biological and Health Sciences, ed. , Milton and Tsokos, McGraw-Hill Book Co. , 1983) . In addition, the optic density (O.D.) of reactive bands in the WB results was recorded by densitometer scanning (Model 620, BioRad Laboratories, Richmond, CA) and scored one plus to three plus according to a panel of standard reactivities which were tested side by side with the test samples in WB assays. Anti-RP-IIB antibody reactivities of a standard PCR-confirmed HTLV-II serum were tested at 1:200, 1:2,000 and 1:10,000 dilutions. For all that had a visible band, the scoring was as follows. A serum sample having reactivity equal to or lower than that of the standard serum at 1:10,000 dilution, the score was one plus; for serum reactivity equal to the standard serum at 1:2,000 or between those of the standard serum at 1:10,000 and 1:2,000, the score was two plus; and the score was three plus when a sample had antibody reactivity greater than that of the standard serum at 1:2000 dilution. This data is also summarized in Table 4.

Twenty-four of 27 (89%) HTLV-II serum samples had two or three plus seroreactivity to RP-IIB while, among those HTLV-I positive samples cross-reacting to RP-IIB,

SUBSTITUTE SHEET only 1 of 14 (7%) had two plus reactivity. This was also reflected by the significant difference in the means of O.D. from densitometer scanning for the above two groups, i.e., 1.19 ± 0.11 for HTLV-II samples and 1.04 ± 0.05 for HTLV-I samples (t-test, p<0.05); Statistical Methods in the Biological and Health Sciences, ed. , Milton and Tsokos, McGraw-Hill Book Co., 1983).

Other Embodiments Antibody reactivity may be recorded using any of a number of immunoassays well known to those skilled in the art. The antigenic peptide can be radio-labelled by conventional methods for use in radioimmunoassay, with fluorescein for fluorescent immunoassay, with enzyme for enzyme immunoassay, or with biotin for biotin-avidin linked assays, it can be employed, labelled or unlabelled as desired, in competitive immunoassays, as well as in double antibody assays or other assays. The antigenic peptide can also be immobilized on some solid phase, such as an insoluble resin (e.g., a nitrocellulose filter) or a microtiter well, and detection of the HTLV antibody carried out by measuring binding of the antibody to the solid phase. Solid phases may also include latex particles, which, when coated with the antigenic peptide and subjected to reactive antibody, will agglutinate. Other examples of solid phases to which the antigenic peptide may be attached include, without limitation, test tubes, vials, titration wells, and the like. Antibody may be detected by double antibody techniques or Protein- A dependent techniques. The diagnostic methodologies of the invention are incorporated into test kits. Such kits are compartmentalized; the first compartment includes one or more of the antigenic peptides of the invention, in detectably labelled form (e.g., any of the forms above) or immobilized on a solid phase (e.g., those listed above) . The second container may include elements necessary for detection of the label on the antigen (e.g., chromogenic substrates) or, alternatively, a second antibody (e.g. , monoclonal or polyclonal anti IgG) directed to the antibody in the biological sample which is specific for the HTLV-encoded antigen. Particular examples of such immunoassays are described in Gallo et al. (U.S. Patent No. 4,520,113, hereby incorporated by reference) . Any biological specimen may be tested, however, of particular interest is the screening of blood to ascertain whether such samples are contaminated with HTLV-I or HTLV-II or both.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Essex, Myron E. Chen, Yi-Ming A.

(11) TITLE OF INVENTION: SPECIFIC DETECTION OF ANTIBODIES TO HUMAN T-CELL LEUKEMIA VIRUSES

(iii) NUMBER OF SEQUENCES:

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Fish & Richardson

(B) STREET: One Financial Center

(C) CITY: Boston

(D) STATE: Massachusetts

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(v) COMPUTER READABLE FORM:

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(D) SOFTWARE: WordPerfect (Version 5.0)

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: N/A

(B) FILING DATE: October 26, 1990

(C) CLASSIFICATION: N/A

(vii) PRIOR APPLICATION DATA:

SUBSTITUTE SHEET Prior applications total, including application described below:

(A) APPLICATION NUMBER: 0

(B) FILING DATE: 0

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Freeman, John W.

(B) REGISTRATION NUMBER: 29,066

(C) REFERENCE/DOCKET #: 00379-009001

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (617) 542-5070

(B) TELEFAX: (617) 542-8906

(C) TELEX: 200154

TOTAL NUMBER OF SEQUENCES TO BE LISTED:

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2486

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(E) NAME: SEQ ID NO. : 1

(ii) SEQUENCE DESCRIPTION FOR SEQUENCE ID NUMBER: 1

TGACAATGGC GACTAGCCTC CCAAGCCAGC CACCCAGGGC GAGTCATCGA CCCAAAAGGT 60 CAGACCGTCT CACACAAACA ATCCCAAGTA AAGGATCTGA CGTCTCCCCC TTTTTTTAGG 120

AACTGAAACC ACGGCCCTGA CGTCCCTCCC CCCTAGGAAC AGGAACAGCT CTCCAGAAAA 180

AAATAGACCT CACCCTTACC CACTTCCCCT AGCGCTGAAA AACAAGGCTC TGACGATTAC 240

CCCCTGCCCA TAAAATTTGC CTAGTCAAAA TAAAAGATGC CGAGTCTATA AAAGCGCAAG 300

GACAGTTCAG GAGGTGGCTC GCTCCCTCAC CGACCCTCTG GTCACGGAGA CTCACCTTGG 360

GGATCCATCC TCTCCAAGCG GCCTCGGTTG AGACGCCTTC CGTGGGACCG TCTCCCGGCC 420

TCGGCACCTC CTGAACTGCT CCTCCCAAGG TAAGTCTCCT CTCAGGTCGA GCTCGGCTGC 480

CCCTTAGGTA GTCGCTCCCC GAGGGTCTTT AGAGACACCC GGGTTTCCGC CTGCGCTCGG 540

CTAGACTCTG CCTTAAACTT CACTTCCGCG TTCTTGTCTC GTTCTTTCCT CTTCGCCGTC 600

ACTGAAAACG AAACCTCAAC GCCGCCCTCT TGGCAGGCGT CCCGGGGCCA ACATACGCCG 660

TGGAGCGCAG CAAGGGCTAG GGCTTCCTGA ACCTCTCCGG GAGAGGTCTA TTGCTATAGG 720

CAGGCCCGCC CTAGGAGCAT TGTCTTCCCG GGGAAGACAA ACAATTGGGG GCTCGTCCGG 780

GATTTGAATT CCTCCATTCT CACATT 806

ATG GGA CAA ATC CAC GGG CTT TCC CCA ACT CCA ATA CCC AAA GCC CCC 854 Met Gly Gin lie His Gly Leu Ser Pro Thr Pro lie Pro Lys Ala Pro

5 10 15

AGG GGG CTA TCA ACC CAC CAC TGG CTT AAC TTT CTC CAG GCT GCT TAC 902 Arg Gly Leu Ser Thr His His Trp Leu Asn Phe Leu Gin Ala Ala Tyr 20 25 30

CGC TTG CAG CCT AGG CCC TCC GAT TTC GAC TTC CAG CAG CTA CGA CGC 950 Arg Leu Gin Pro Arg Pro Ser Asp Phe Asp Phe Gin Gin Leu Arg Arg 35 40 45

TTT CTA AAA CTA GCC CTT AAA ACG CCC ATT TGG CTA AAT CCT ATT GAC 998 Phe Leu Lys Leu Ala Leu Lys Thr Pro lie Trp Leu Asn Pro He Asp 50 55 60

TAC TCG CTT TTA GCT AGC CTT ATC CCC AAG GGA TAT CCA GGA AGG GTG 1046

Tyr Ser Leu Leu Ala Ser Leu He Pro Lys Gly Tyr Pro Gly Arg Val

65 70 75 80

GTA GAG ATT ATA AAT ATC CTT GTC AAA AAT CAA GTC TCC CCT AGC GCC 1094 Val Glu He He Asn He Leu Val Lys Asn Gin Val Ser Pro Ser Ala 85 90 95

CCC GCC GCC CCA GTT CCG ACA CCT ATC TGC CCT ACT ACT ACT CCT CCG 1142 Pro Ala Ala Pro Val Pro Thr Pro He Cys Pro Thr Thr Thr Pro Pro 100 105 110

CCA CCT CCC CCC CCT TCC CCG GAG GCC CAT GTT CCC CCC CCT TAC GTG 1190 Pro Pro Pro Pro Pro Ser Pro Glu Ala His Val Pro Pro Pro Tyr Val 115 120 125

GAA CCC ACC ACC ACG CAA TGC TTC CCT ATC TTA CAT CCC CCA GGA GCC 1238

SUBSTITUTE SHEET Glu Pro Thr Thr Thr Gin Cys Phe Pro He Leu His Pro Pro Gly Ala 130 135 140

CCC TCA GCT CAT AGG CCC TGG CAG ATG AAA GAC TTA CAG GCC ATC AAG 1286 Pro Ser Ala His Arg Pro Trp Gin Met Lys Asp Leu Gin Ala He Lys 145 150 155 160

CAG GAG GTC AGC TCC TCT GCT CTT GGC AGC CCC CAG TTC ATG CAG ACC 1334 Gin Glu Val Ser Ser Ser Ala Leu Gly Ser Pro Gin Phe Met Gin Thr 165 170 175

CTC CGG CTG GCG GTA CAA CAG TTT GAC CCC ACC GCC AAG GAC TTA CAA 1382 Leu Arg Leu Ala Val Gin Gin Phe Asp Pro Thr Ala Lys Asp Leu Gin 180 185 190

GAT CTC CTC CAG TAC CTA TGC TCC TCC CTC GTA GTT TCC TTA CAC CAT 1430 Asp Leu Leu Gin Thr Leu Cys Ser Ser Leu Val Val Ser Leu His His 195 200 205

CAG CAG CTT AAC ACA CTA ATT ACC GAG GCT GAG ACC CGC GGG ATG ACA 1478 Gin Gin Leu Asn Thr Leu He Thr Glu Ala Glu Thr Arg Gly Met Thr 210 215 220

GGC TAC AAC CCC ATG GCA GGG CCC CTA AGA ATG CAG GCT AAT AAC CCC 1526 Gly Tyr Asn Pro Met Ala Gly Pro Leu Arg Met Gin Ala Asn Asn Pro 225 230 235 240

GCC CAG CAA GGT CTT AGA CGG GAG TAC CAG AAT CTT TGG CTG GCT GCT 1574 Ala Gin Gin Gly Leu Arg Arg Glu Thr Gin Asn Leu Trp Leu Ala Ala 245 250 255

TTC TCC ACC CTG CCA GGC AAT ACC CGT GAC CCC TCT TGG GCA GCT ATC 1622 Phe Ser Thr Leu Pro Gly Asn Thr Arg Asp Pro Ser Trp Ala Ala He 260 265 270

CTA CAG GGG CTG GAG GAA CCC TAT TGC GCG TTC GTA GAG CGC CTT AAC 1670 Leu Gin Gly Leu Gly Glu Pro Tyr Cys Ala Phe Val Glu Arg Leu Asn 275 280 285

GTG GCC CTT GAC AAC GGC CTC CCC GAG GGT ACC CCC AAA GAG CCC ATC 1718 Val Ala Leu Asp Asn Gly Leu Pro Glu Gly Thr Pro Lys Glu Pro He 290 295 300

TTA CGT TCC CTA GCG TAC TCA AAC GCC AAC AAA GAA TGC CAA AAA ATC 1766 Leu Arg Ser Leu Ala Tyr Ser Asn Ala Asn Lys Glu Cys Gin Lys He 305 310 315 320

TTA CAA GCC CGC GGA CAC ACT AAC AGC CCC CTT GGG GAG ATG CTC CGG 1814 Leu Gin Ala Arg Gly His Thr Asn Ser Pro Leu Gly Glu Met Leu Arg 325 330 335

ACA TGT CAG GCG TGG ACA CCC AAG GAC AAA ACC AAG GTC CTT GTG GTC 1862 Thr Cys Gin Ala Trp Thr Pro Lys Asp Lys Thr Lys Val Leu Val Val

SUBSTITUTE SHEET 340 345 350

CAA CCA CGG AGG CCC CCC CCC ACA CAG CCC TGC TTT CGT TGT GGC AAG 1910 Gin Pro Arg Arg Pro Pro Pro Thr Gin Pro Cys Phe Arg Cys Gly Lys 355 360 365

GTA GGA CAC TGG AGT CGG GAC TGT ACC CAG CCA CGC CCC CCT CCT GGC 1958 Val Gly His Trp Ser Arg Asp Cys Thr Gin Pro Arg Pro Pro Pro Gly 370 375 380

CCC TGC CCC CTA TGC CAA GAT CCT TCT CAC TGG AAA AGG GAC TGC CCA 2006 Pro Cys Pro Leu Cys Gin Asp Pro Ser His Trp Lys Arg Asp Cys Pro 385 390 395 400

CAA CTC AAA CCC CCT CAG GAG GAA GGG GAA CCC CTC CTG TTG GAT CTC 2054 Gin Leu Lys Pro Pro Gin Glu Glu Gly Glu Pro Leu Leu Leu Asp Leu 405 410 415

CCT TCC ACC TCA GGC ACT ACT GAG GAA AAA AAC TCC TTA AGG GGG GAG 2102 Pro Ser Thr Ser Gly Thr Thr Glu Glu Lys Asn Ser Leu Arg Gly Glu 420 425 430

Gly Lys Lys Leu Leu Lys Gly Gly

5

ATC TAA TCT CCC CCC ATC CCG ATC AAG ACA TCT CGA TAC TCC CAC TCA 2150 He

Asp Leu He Ser Pro His Pro Asp Gin Asp He Ser He Leu Pro Leu 10 15 20

TCC CCC TGC GGC AGC AAC AGC AAC CAA TTC TAG GGG TCC GGA TCT CCG 2198 He Pro Leu Arg Gin Gin Gin Gin Pro He Leu Gly Val Arg He Ser 25 30 35 40

TTA TGG GAC AAA CAC CTC AGC CTA CCC AAG CGC TAC TTG ACA CAG GAG 2246

His Arg Ser

Val Met Gly Gin Thr Pro Gin Pro Thr Gin Ala Leu Leu Asp Thr Gly 45 50 55

CCG ACC TTA CGG TTA TAC CCC AGA CAC TCG TGC CCG GGC CGG TAA AGC 2294 Arg Pro Tyr Gly Tyr Thr Pro Asp Thr Arg Ala Arg Ala Gly Lys Ala 5 10 15

Ala Asp Leu Thr Val He Pro Gin Thr Leu Val Pro Gly Pro Val Lys 60 65 70

TCC ACG ACA CCC TGA TCC TAG GCG CCA GTG GGC AAA CCA ACA CCC AGT 2342 Pro Arg His Pro Asp Pro Arg Arg Gin Trp Ala Asn Gin His Pro Val 20 25 30 35

Leu His Asp Thr Leu He Leu Gly Ala Ser Gly Gin Thr Asn Thr Gin 75 80 85

TCA AAC TCC TCC AAA CCC CCC TAC ACA TAT TCT TGC CCT TCC GAA GGT 2390

SUBSTITUTE SHEET Gin Thr Pro Pro Asn Pro Pro Thr His He Leu Ala Leu Pro Lys Val 40 45 50

Phe Lys Leu Leu Gin Thr Pro Leu His He Phe Leu Pro Phe Arg Arg 90 95 100

CCC CCG TTA TCC TTT CCT CCT GCC TCT TAG ACA CCC ACA ACA AAT GGA 2438 Pro Arg Tyr Pro Phe Leu Leu Pro Leu Arg His Pro Gin Gin Met Asp 55 60 65

Ser Pro Val He Leu Ser Ser Cys Leu Leu Asp Thr His Asn Lys Trp 105 110 115 120

CCA TCA TTG GAA GGG ACG CCC TAC AAC AAT GCC AGG GGC TTC TAT ACC 2486 His His Trp Lys Gly Arg Pro Thr Thr Met Pro Gly Ala Ser He Pro 70 75 80

Thr He He Gly Arg Asp Ala Leu Gin Gin Cys Gin Gly Leu Leu Tyr 125 130 135

TCC CAG ACG ACC CCA GCC CCC ACC AAT TGC TGC CAA TAG CCA CTC CAA 2534 Pro Arg Arg Pro Gin Pro Pro Pro He Ala Ala Asn Ser His Ser Lys 85 90 95

Leu Pro Asp Asp Pro Ser Pro His Gin Leu Leu Pro He Ala Thr Pro 140 145 150

ACA CCA TAG GCC TCG AAC ACC TTC CCC CAC CTC CCC AAG TGG ACC AAT 2582 His His Arg Pro Arg Thr Pro Ser Pro Thr Ser Pro Ser Gly Pro He 100 105 110

Asn Thr He GLy Leu Glu His Leu Pro Pro Pro Pro Gin Val Asp Gin 155 160 165

TTC CTT TAA ACC TGA GCG CCT CCA GGC CTT AAA TGA CCT GGT CTC CAA 2630 Ser Phe Lys Pro Glu Arg Leu Gin Ala Leu Asn Asp Leu Val Ser Lys 115 120 125 130

Phe Pro Leu Asn Leu Ser Ala Ser Arg Pro 170 175

GGC CCT GGA GGC TGG TCA CAT TGA ACC ATA CTC AGG ACC AGG CAA TAA 2678 Ala Leu Glu Ala Gly His He Glu Pro Tyr Ser Gly Pro.Gly Asn Asn 135 140 145

CCC CGT CTT CCC CGT TAA AAA ACC AAA TGG TAA ATG GAG GTT CAT TCA 2726 Pro Val Phe Pro Val Lys Lys Pro Asn Gly Lys Trp Arg Phe He His 150 155 160

TGA CCT AAG AGC CAC CAA TGC CAT TAC TAC CAC CCT CAC CTC TCC TTC 2774 Asp Leu Arg Ala Thr Asn Ala He Thr Thr Thr Leu Thr Ser Pro Ser 165 170 175

CCC AGG GCC CCC CGA TCT CAC TAG CCT ACC GAC AGC CTT ACC CCA CCT 2822 Pro Gly Pro Pro Asp Leu Thr Ser Lue Pro Thr Ala Leu Pro His Leu 180 185 190

ACA GAC CAT AGA TCT TAC TGA CGC CTT TTT CCA AAT CCC CCT CCC CAA 2870 Gin Thr He Asp Leu Thr Asp Ala Phe Phe Gin He Pro Leu Pro Lys 195 200 205 210

GCA GTA CCA GCC ATA CTT CGC CTT CAC CAT TCC CCA GCC ATG TAA CTA 2918 Gin Tyr Gin Pro Tyr Phe Ala Phe Thr He Pro Gin Pro Cys Asn Tyr 215 220 225

TGG CCC CGG GAC CAG ATA TGC ATG GAC TGT CCT TCC ACA GGG GTT TAA 2966 Gly Pro Gly Thr Arg Tyr Ala Trp Thr Val Leu Pro Gin Gly Phe Lys 230 235 240

AAA CAG CCC CAC CCT CTT CGA ACA ACA ATT AGC AGC CGT CCT CAA CCC 3014 Asn Ser Pro Thr Leu Phe Glu Gin Gin Leu Ala Ala Val Leu Asn Pro 245 250 255

CAT GAG GAA AAT GTT TCC CAC ATC GAC CAT TGT CCA ATA CAT GGA TGA 3062 Met Arg Lys Met Phe Pro Thr Ser Thr He Val Gin Tyr Met Asp Asp 260 265 270

CAT ACT TTT AGC CAG CCC CAC CAA TGA GGA ATT ACA ACA ACT CTC CCA 3110 He Leu Leu Ala Ser Pro Thr Asn Glu Glu Leu Gin Gin Leu Ser Gin 275 280 285 290

GCT AAC CCT CCA GGC ACT GAC CAC GCA TGG CCT TCC AAT TTC CCA GGA 3158 Leu Thr Leu Gin Ala Leu Thr Thr His Gly Leu Pro He Ser Gin Glu 295 300 305

AAA AAC ACA ACA AAC CCC AGG CCA AAT ACG CTT CTT AGG ACA GGT CAT 3206 Lys Thr Gin Gin Thr Pro Gly Gin He Arg Phe Lue Gly Gin Val He 310 ^• 315 320

CTC CCC TAA TCA CAT TAC ATA TGA GAG TAC CCC TAC TAT TCC CAT AAA 3254 Ser Pro Asn His He Thr Tyr Glu Ser Thr Pro Thr He Pro He Lys 325 330 335

ATC CCA ATG GAC ACT CAC TGA ATT ACA AGT TAT CCT AGG AGA GAT CCA 3302 Ser Gin Trp Thr Leu Thr Glu Leu Gin Val He Leu Gly Glu He Gin 340 345 350

GTG GGT CTC TAA AGG AAC ACC CAT CCT TCG CAA ACA CCT ACA ATC CCT 3350 Trp Val Ser Lys Gly Thr Pro He Leu Arg Lys His Leu Gin Ser Leu 355 360 365 370

ATA TTC TGC CCT TCA CGG GTA CCG GGA CCC AAG AGC TTG TAT CAC CCT 3398 Tyr Ser Ala Leu His Gly Tyr Arg Asp Pro Arg Ala Cys He Thr Leu 375 380 385

CAC CCC ACA ACA ACT CCA TGC GTT ACA TGC CAT TCA ACA AGC TCT ACA 3446 Thr Pro Gin Gin Leu His Ala Leu His Ala He Gin Gin Ala Leu Gin 390 395 400

ACA TAA CTG CCG TGG CCG CCT CAA CCC CGC CCT ACC TCT CCT TGG CCT 3494 His Asn Cys Arg Gly Arg Leu Asn Pro Ala Leu Pro Leu Leu Gly Leu 405 410 415

SUBSTITUTE SHEET CAT CTC GTT AAG TAC ATC TGG TAC AAC ATC TGT CAT CTT TCA ACC CAA 3542 He Ser Leu Ser Thr Ser Gly Thr Thr Ser Val He Phe Gin Pro Lys 420 425 430

GCA AAA TTG GCC CCT GGC TTG GCT CCA CAC CCC CCA CCC TCC GAC CAG 3590 Gin Asn Trp Pro Leu Ala Trp Leu His Thr Pro His Pro Pro Thr Ser 435 440 445 450

TTT ATG TCC TTG GGG TCA CCT ACT GGC CTG CAC CAT CTT AAC TCT AGA 3638 Leu Cys Pro Trp Gly His Leu Leu Ala Cys Thr He Leu Thr Leu Asp 455 460 465

CAA ATA TAC CCT ACA ACA TTA TGG CCA GCT CTG CCA ATC TTT CCA CCA 3686 Lys Tyr Thr Leu Gin His Tyr Gly Gin Leu Cys Gin Ser Phe His His 470 475 480

CAA CAT GTC AAA GCA AGC CCT TTG CGA CTT CCT GAG GAA CTC CCC TCA 3734 Asn Met Ser Lys Gin Ala Leu Cys Asp Phe Leu Arg Asn Ser Pro His 485 490 495

TCC AAG TGT CGG CAT CCT CAT TCA CCA CAT GGG TCG ATT CCA TAA CCT 3782 Pro Ser Val Gly He Leu He His His Met Gly Arg Phe His Ash Leu 500 505 510

TGG CAG CCA ACC GTC TGG TCC GTG GAA GAC TCT CTT ACA CCT CCC AAC 3830 Gly Ser Gin Pro Ser Gly Pro Trp Lys Thr Leu Leu His Leu Pro Thr 515 520 525 530

CCT TCT CCA GGA ACC ACG ACT CCT CAG GCC AAT TTT CAC CCT CTC CCC 3878 Leu Leu Gin Glu Pro Arg Leu Leu Arg Pro He Phe Thr Leu Ser Pro 535 540 545

CGT CGT GCT TGA CAC GGC CCC CTG CCT TTT TTC CGA TGG CTC CCC TCA 3926 Val Val Leu Asp Thr Ala Pro Cys Leu Phe Ser Asp Gly Ser Pro Gin 550 555 560

AAA GGC AGC GTA CGT TCT CTG GGA CCA GAC TAT CCT TCA ACA GGA CAT 3974 Lys Ala Ala Tyr Val Leu Trp Asp Gin Thr He Leu Gin Gin Asp He 565 570 575

CAC TCC CCT GCC CTC TCA CGA AAC ACA TTC CGC ACA AAA GGG GGA GCT 4022 Thr Pro Leu Pro Ser His Glu Thr His Ser Ala Gin Lys Gly Glu Leu 580 585 590

CCT TGC ACT TAT CTG TGG ACT ACG TGC TGC CAA GCC ATG GCC TTC CCT 4070 Leu Ala Leu He Cys Gly Leu Arg Ala Ala Lys Pro Trp Pro Ser Leu 595 600 605 610

TAA CAT CTT TTT AGA CTC TAA ATA TTT AAT CAA ATA CCT ACA TTC CCT 4118 Asn He Phe Leu Asp Ser Lys Tyr Leu He Lys Tyr Leu His Ser Leu 615 620 625

SUBSTITUTE SHEET CGC CAT TGG GGC CTT CCT CGG CAC TTC CTC CCA TCA AAC CCT CCA GGC 4166 Ala He Gly Ala Phe Leu Gly Thr Ser Ala His Gin Thr Leu Gin Ala 630 635 640

GGC CTT GCC ACC CCT ACT GCA GGG CAA GAC CAT CTA CCT CCA CCA TGT 4214 Ala Leu Pro Pro Leu Leu Gin Gly Lys Thr He Tyr Leu His His Val 645 650 655

CCG CAC CCA CAC CAA CCT CCC CGA CCC AAT TTC CAC CTT CAA TGA ATA 4262 Arg Ser His Thr Asn Leu Pro Asp Pro He Ser Thr Phe Asn Glu Tyr 660 665 670

CAC AGA CTC CCT TAT CTT AGC TCC CCT TGT TCC CCT GAC GCC CCA AGG 4310 Thr Asp Ser Leu He Leu Ala Pro Leu Val Pro Leu Thr Pro Gin Gly 675 680 685 690

CCT CCA CGG C 4320

Leu His Gly

CTC ACC CAT TGC AAT CAA AGG GCT CTA GTC TCT TTT GGC GCC ACA CCA 4368 Leu Thr His Cys Asn Gin Arg Ala Leu Val Ser Phe Gly Ala Thr Pro 695 700 705

AGG GAA GCC AAG TCC CTT GTA CAG ACT TGC CAT ACC TGT CAA ACC ATC 4416 Arg Glu Ala Lys Ser Leu Val Gin Thr Cys His Thr Cys Gin Thr He 710 715 720 725

AAC TCA CAA CAT CAT ATG CCT CGA GGG TAC ATT CGC CGG GGC CTC TTG 4464 Asn Ser Gin His His Met Pro Arg Gly Tyr He Arg Arg Gly Leu Leu 730 735 740

CCC AAC CAC ATA TGG CAA GGT GAT GTA ACC CAT TAT AAG TAC AAA AAA 4512 Pro Asn His He Trp Gin Gly Asp Val Thr His Tyr Lys Tyr Lys Lys 745 750 755

TAC AAA TAC TGC CTC CAC GTC TGG GTA GAC ACC TTC TCC GGT GCG GTT 4560 Tyr Lys Tyr Cys Leu His Val Trp Val Asp Thr Phe Ser Gly Ala Val 760 765 770

TCC GTC TCC TGT AAA AAG AAA GAA ACC AGC TGT GAG ACT ATC AGC GCC 4608 Ser Val Ser Cys Lys Lys Lys Glu Thr Ser Cys Glu Thr He Ser Ala 775 780 785

GTT CTT CAG GCC ATT TCC CTC CTA GGG AAA CCA CTC CAC ATT ACC ACA 4656 Val Leu Gin Ala He Ser Leu Leu Gly Lys Pro Leu His He Asn Thr 790 795 800 805

GAT AAT GGG CCA GCC TTC CTA TCA CAA GAA TTC CAG GAG TTT TGT ACC 4704 Asp Asn Gly Pro Ala Phe Leu Ser Gin Glu Phe Gin Glu Phe Cys Thr 810 815 820

TCC TAT CGC ATC AAG CAT TCT ACC CAT ATA CCA TAC AAC CCC ACC AGC 4752

SUBSTITUTE SHEET Ser Tyr Arg He Lys His Ser Thr His He Pro Tyr Asn Pro Ser Gly 825 830 835

TCA GGC CTG GTC GAG AGA ACC AAT GGT GTA ATC AAA AAC TTA CTA AAT 4800 Leu Val Glu Arg Thr Asn Gly Val He Lys Asn Leu Leu Asn Leu Asn 840 845 850

AAA TAT CTA CTA GAC TGT CCT AAC CTT CCC CTA GAC AAT GCC ATT CAC 4848 Lys Tyr Leu Leu Asp Cys Pro Asn Leu Pro Leu Asp Asn Ala He His 855 860 865

AAA GCC CTT TGG ACT CTC AAT CAG CTA AAT GTC ATG AAC CCC AGT GGT 4896 Lys Ala Leu Trp Thr Leu Asn Gin Leu Asn Val Met Asn Pro Ser Gly 870 875 880 885

AAA ACC CGA TGG CAA ATC CAC CAC AGT CCT CCA CTA CCA CCC ATT CCT 4944 Lys Thr Arg Trp Gin He His His Ser Pro Pro Leu Pro Pro He Pro 890 895 900

GAA GCC TCT ACC CCT CCC AAA CCA CCT CCC AAA TGG TTC TAT TAT AAA 4992 Glu Ala Ser Thr Pro Pro Lys Pro Pro Pro Lys Trp Phe Tyr Tyr Lys 905 910 915

CTC CCC GGC CTT ACC AAT CAG CGG TGG AAA GGT CCA TTG CAA TCC CTC 5040 Leu Pro Gly Leu Thr Asn Gin Arg Trp Lys Gly Pro Leu Gin Ser Leu 920 925 930

CAG GAA GCG GCC GGG GCA GCC TTG CTC TCC ATA GAC GGC TCC CCC CGG 5088 Gin Glu Ala Ala Gly Ala Ala Leu Leu Ser He Asp Gly Ser Pro Arg 935 940 945

TGG ATC CCG TGG CGA TTC CTG AAA AAA GCT GCA TGC CCA AGA CCA GAC 5136 Trp He Pro Trp Arg Phe Leu Lys Lys Ala Ala Cys Pro Arg Pro Asp 950 955 960 965

GCC AGC GAA CTC GCC GAG CAC GCC GCA ACA GAC CAC CAA CAC CAT GGGT 5185 Ala Ser Glu Leu Ala Glu His Ala Ala Thr Asp His Gin His His Gly 970 975 980

Met Gly

AAT GTT TTC TTC CTA CTT TTA TTC AGT CTC ACA CAT TTT CCA CTA GCC 5233 Asn Val Phe Phe Leu Leu Leu Phe Ser Leu Thr His Phe Pro Leu Ala 5 10 15

CAG CAG AGC CGA TGC ACA CTC ACG ATT GGT ATC TCC TCC TAC CAC TCC 5281 Gin Gin Ser Arg Cys Thr Leu Thr He Gly He Ser Ser Tyr His Ser 20 25 30

AGC CCC TGT AGC CCA ACC CAA CCC GTC TGC ACG TGG AAC CTC GAC CTT 5329 Ser Pro Cys Ser Pro Thr Gin Pro Val Cys Thr Trp Asn Leu Asp Leu 35 40 45 50

SUBSTITUTE SHEET AAT TCC CTA ACA ACG GAC CAA CGA CTA CAC CCC CCT TGC CCT AAC CTA 5377 Asn Ser Leu Thr Thr Asp Gin Arg Leu His Pro Pro Cys Pro Asn Leu 55 60 65

ATT ACT TAC TCT GGC TTC CAT AAC ACT TAT TCC TTA TAC TTA TTC CCA 5425 He Thr Tyr Ser Gly Phe His Lys Thr Tyr Ser Leu Tyr Leu Phe Pro 70 75 80

CAT TGG ATA AAA AAG CCA AAC AGA CAG GGC CTA GGG TAC TAC TCG CCT 5473 His Trp He Lys Lys Pro Asn Arg Gin Gly Leu Gly Tyr Tyr Ser Pro 85 90 95

TCC TAC AAT GAC CCT TGC TCG CTA CAA TGC CCC TAC TTG GGC TGC CAA 5521 Ser Tyr Asn Asp Pro Cys Ser Leu Gin Cys Pro Tyr Leu Gly Cys Gin 100 105 110

GCA TGG ACA TCC GCA TAC ACG GGC CCC GTC TCC AGT CCA TCC TGG AAG 5569 Ala Trp Thr Ser Ala Tyr Thr Gly Pro Val Ser Ser Pro Ser Trp Lys 115 120 125 130

TTT CAT TCA GAT GTA AAT TTC ACC CAG GAA GTC AGC CAA GTG TCC CTT 5617 Phe His Ser Asp Val Asn Phe Thr Gin Glu Val Ser Gin Val Ser Leu 135 140 145

CGA CTA CAC TTC TCT AAG TGC GGC TCC TCC ATG ACC CTC CTA GTA GAT 5665 Arg Leu His Phe Ser Lys Cys Gly Ser Ser Met Thr Leu Leu Val Asp 150 155 160

GCC CCT GGA TAT GAT CCT TTA TGG TTC ATC ACC TCA GAA CCC ACT CAG 5713 Ala Pro Gly Tyr Asp Pro Leu Trp Phe He Thr Ser Glu Pro Thr Gin 165 170 175

CCT CCA CCA ACT TCT CCC CCA TTG GTC CAT GAC TCC GAC CTT GAA CAT 5761 Pro Pro Pro Thr Ser Pro Pro Leu Val His Asp Ser Asp Leu Glu His 180 185 190

GTC CTA ACC CCC TCC ACG TCC TGG ACG ACC AAA ATA CTC AAA TTT ATC 5809 Val Leu Thr Pro Ser Thr Ser Trp Thr Thr Lys He Leu Lys Phe He 195 200 205 210

CAG CTG ACC TTA CAG AGC ACC AAT TAC TCC TGC ATG GTT TGC GTG GAT 5857 Gin Leu Thr Leu Gin Ser Thr Asn Tyr Ser Cys Met Val Cys Val Asp 215 220 225

AGA TCC AGC CTC TCA TCC TGG CAT GTA CTC TAC ACC CCC AAC ATC TCC 5905 Arg Ser Ser Leu Ser Ser Trp His Val Leu Tyr Thr Pro Asn He Ser 230 235 240

ATT CCC CAA CAA ACC TCC TCC CGA ACC ATC CTC TTT CCT TCC CTT GCC 5953 He Pro Gin Gin Thr Ser Ser Art Thr He Leu Phe Pro Ser Leu Ala 245 250 255

CTG CCC GCT CCT CCA TCC CAA CCC TTC CCT TGG ACC CAT TGC TAC CAA 6001

SUBSTITUTE SHEET Leu Pro Ala Pro Pro Ser Gin Pro Phe Pro Trp Thr His Cys Tyr Gin 260 265 270

CCT CGC CTA CAG GCA ATA ACA ACA GAT AAC TGC AAC AAC TCC ATT ATC 6049 Pro Arg Leu Gin Ala He Thr Thr Asp Asn Cys Asn Asn Ser He He 275 280 285 290

CTC CCC CCT TTT TCC CTC GCT CCC GTA CCT CCT CCG GCG ACA AGA CGC 6097 Leu Pro Pro Phe Ser Leu Ala Pro Val Pro Pro Pro Ala Thr Arg Arg 295 300 305

CGC CGT GCC GTT CCA ATA GCA GTG TGG CTT GTC TCC GCC CTA GCG GCO 6145 Art Arg Ala Val Pro He Ala Val Trp Leu Val Ser Ala Leu Ala Ala 310 315 320

GGA ACA GGT ATC GCT GGT GGA GTA ACA GGC TCC CTA TCT CTG GCT TCC 6193 Gly Thr Gly He Ala Gly Gly Val Thr Gly Ser Leu Ser Leu Ala Ser 325 330 335

AGT AAA AGC CTT CTC CTC GAG GTT GAC AAA GAC ATC TCC CAC CTT ACC 6241 Ser Lys Ser Leu Leu Leu Glu Val Asp Lys Asp He Ser His Leu Thr 340 345 350

CAG GCC ATA GTC AAA AAT CAT CAA AAC ATC CTC CGG GTT GCA CAG TAT 6289 Gin Ala He Val Lys Asn His Gin Asn He Leu Arg Val Ala Gin Tyr 355 360 365 370

GCA GCC CAA AAT AGA CGA GGA TTA GAC CTC CTA TTC TGG GAA CAA GGG 6337 Ala Ala Gin Asn Arg Arg Gly Leu Asp Leu Leu Phe Trp Glu Gin Gly 375 380 385

GGT TTG TGC AAG GCC ATA CAG GAG CAA TGT TGC TTC CTC AAC ATC AGT 6385 Gly Leu Cys Lys Ala He Gin Glu Gin Cys Cys Phe Leu Asn He Ser 390 395 400

AAC ACT CAT GTA TCC GTC CTC CAG GAA CGG CCC CCT CTT GAA AAA CGT 6433 Asn Thr His Val Ser Val Leu Gin Glu Arg Pro Pro Leu Glu Lys Arg 405 410 415

GTC ATC ACC GGC TGG GGA CTA AAC TGG GAT CTT GGA CTG TCC CAA TGG 6481 Val He Thr Gly Trp Gly Leu Asn Trp Asp Leu Gly Leu Ser Gin Trp 420 425 430

GCA CGA GAA GCC CTC CAG ACA GGC ATA ACC ATT CTC GCT CTA CTC CTC 6529 Ala Arg Glu Ala Leu Gin Thr Gly He Thr He Leu Ala Leu Leu Leu 435 440 445 450

CTC GTC ATA TTG TTT GGC CCC TGT ATC CTC CGC CAA ATC CAG GCC CTT 6577 Leu Val He Leu Phe Gly Pro Cys He Leu Arg Gin He Gin Ala Leu 455 460 465

SUBSTITUTE SHEET CCA CAG CGG TTA CAA AAC CGA CAT AAC CAG TAT TCC CTT ATC AAC CCA 6625 Pro Gin Arg Leu Gin Asn Arg His Asn Gin Tyr Ser Leu He Asn Pro 470 475 480

GAA ACC ATG CTA 6637 Glu Thr Met Leu

TAATAGACCT GCTAGCTTCT GCAGCAAATC CCCTAGGTTC GTCCCCCTAC CATTGACCCA 6697

TCCACAGTCC TCTATACCAG ATGAGTCGCC CCCGATGTCC AGCCCTAACT CGATTCTGAA 6757

TAATTGCCTC AAATAGTTCC TCTAACCCCC GCTCACATTC CTCCCATAGG ACCTTCTTTT 6817

CCCCTTCAGG AAATCCACAT AACCCTGAAG CAAGTCACAA AACCCATCAA AACCCAGGAG 6877

TCCTATACAC TCCAACTGCT GATGCCTTTC TTCCCTCTCC CGGCGCTTTT GATCCTTTTC 6937

CCGCAGGCGC TCCTTTCTGC GCCGCTCCCG CTCCTCACGC TCCTGCAGAA GTTTTAAGAT 6997

CTCCCGCTGC TCCTCCGCCA ACAGTCTCCG ACGAGAGTCT CGCACCTGCT CGCTGACCGA 7057

TCCCGACCCC AGAGGGCGAC CTTTTGCTGT CCTTCTCGGT TCCTCTCCAG GGGGAGGGAC 7117

ACCAGATGTC AGACTCGCCT CTCCCTGGTC TCCTAACGGC AATCTCCTAA AATAGTCTAA 7177

AAAATCACAC ATAA 7191

TTA CAA TCC TGT CTC CTC TCA GCC CAT TTC CTA GGA TTT GGA CAG AGC 7239 Leu Gin Ser Cys Leu Leu Ser Ala His Phe Leu Gly Phe Gly Gin Ser

5 10 15

CTC CTA TAT GGA TAC CCC GTC TAC GTG TTT GGC GAT TGT GTA CAG GCC 7287 Leu Leu Tyr Gly Tyr Pro Val Tyr Val Phe Gly Asp Cys Val Gin Ala 20 25 30

GAT TGG TGT CCC GTC TCA GGT GGT CTA TGT TCC ACC CGC CTA CAT CGA 7335 Asp Trp Cys Pro Val Ser Gly Gly Leu Cys Ser Thr Arg Leu His Arg 35 40 45

CAT GCC CTC CTG GCC ACC TGT CCA GAG CAC CAA CTC ACC TGG GAC CCC 7383 His Ala Leu Leu Ala Thr Cys Pro Glu His Gin Leu Thr Trp Asp Pro 50 55 60

ATC GAT GGA CGC GTT GTC AGC TCT CCT CTC CAA TAC CTT ATC CCT CGC 7431 He Asp Gly Arg Val Val Ser Ser Pro Leu Gin Tyr Leu He Pro Arg 65 70 75 80

CTC CCC TCC TTC CCC ACC CAG AGA ACC TCA AGG ACC CTC AAG GTC CTT 7479 Leu Pro Ser Phe Pro Thr Gin Arg Thr Ser Arg Thr Leu Lys Val Leu 85 90 95

ACC CCT CCC ACC ACT CCT GTC TCC CCC AAG GTT CCA CCT GCC TTC TTT 7527

SUBSTITUTE SHEET Thr Pro Pro Thr Thr Pro Val Ser Pro Lys Val Pro Pro Ala Phe Phe 100 110 115

CAA TCA ATG CGA AAG CAC ACC CCC TAC CGA ATT GGA TGC CTG GAA CCA 7575 Gin Ser Met Arg Lys His Thr Pro Tyr Arg Asn Gly Cys Leu Glu Pro 120 125 130

ACC CTC GGG GAT CAG CTC CCC TCC CTC GCC TTC CCC GAA CCT GGC CTC 7623 Thr Leu Gly Asp Gin Leu Pro Ser Leu Ala Phe Pro Glu Pro Gly Leu 135 140 145

CGT CCC CAA AAC ATC TAC ACC ACC TGG GGA AAA ACC GTA GTA TGC CTA 7671 Arg Pro Gin Asn He Tyr Thr Thr Trp Gly Lys Thr Val Val Cys Leu 150 155 160 165

TAC CTA TAC CAG CTT TCC CCA CCC ATG ACA TGG CCA CTT ATA CCC CAT 7719 Tyr Leu Tyr Gin Leu Ser Pro Pro Met Thr Trp Pro Leu He Pro His 170 175 180

GTC ATA TTC TGC CAC CCC AGA CAA TTA GGA GCC TTC CTC ACC AAG GTC 7767 Val He Phe Cys His Pro Arg Gin Leu Gly Ala Phe Leu Thr Lys Val 185 190 195

CCT CTA AAA CGA TTA GAA GAA CTT CTA TAC AAA ATG TTC CTA CAC ACA 7815 Pro Leu Lys Art Leu Glu Glu Leu Leu Tyr Lys Met Phe Leu His Thr 200 205 210

GGG ACA GTC ATA GTC CTC CCG GAG GAC GAC CTA CCC ACC ACA ATG TTC 7863 Gly Thr Val He Val Leu Pro Glu Asp Asp Leu Pro Thr Thr Met Phe 215 220 225

CAA CCC GTG AGG GCT CCC TGT ATC CAG ACT GCC TGG TGT ACA GGA CTT 7911 Gin Pro Val Arg Ala Pro Cys He Gin Thr Ala Trp Cys Thr Gly Leu 230 235 240 245

CTC CCC TAT CAC TCC ATC TTA ACA ACC CCA GGT CTA ATA TGG ACC TTC 7959 Leu Pro Tyr His Ser He Leu Thr Thr Pro Gly Leu He Trp Thr Phe 250 255 .260

AAT GAC GGC TCA CCA ATG ATT TCC GGC CCT TAC CCC AAA GCA GGG CAG 8007 Asn Asp Gly Ser Pro met He Ser Gly Pro Tyr Pro Lys Ala Gly Gin 265 270 275

CCA TCT TTA GTA GTT CAG TCC TCC CTA TTA ATC TTC GAA AAA TTC GAA 8055 Pro Ser Leu Val Val Gin Ser Ser Leu Leu He Phe Glu Lys Phe Glu 280 285 290

ACC AAA GCC TTC CAT CCC TCC TAT CTA CTC TCT CAT CAG CTT ATA CAA 8103 Thr Lys Ala Phe His Pro Ser Tyr Leu Leu Ser His Gin Leu He Gin 295 300 305

TAC TCC TCC TTC CAT AAC CTT CAC CTT CTA TTC GAT GAA TAC ACC AAC 8151 Tyr Ser Ser Phe His Asn Leu His Leu Leu Phe Asp Glu Tyr Thr Asn

SUB 310 315 320 325

ATC CCT GTC TCT ATT TTA TTT AAT AAA GAA GAG GCG GAT GAC AAT GGC 8199 He Pro Val Ser He Leu Phe Asn Lys Glu Glu Ala Asp Asp Asn Gly 330 335 340

GAC 8202

Asp

TAGCCTCCCG AGCCAGCCAC CCAGGGCGAG TCATCGACCC AAAAGGTCAG ACCGTCTCAC 8262 ACAAACAATC CCAAGTAAAG GCTCTGACGT CTCCCCCTTT TTTTAGGAAC TGAAACCACG 8322 GCCCTGACGT CCCTCCCCCC TAGGAACAGG AACAGCTCTC CAGAAAAAAA TAGACCTCAC 8382

CCTTACCCAC TTCCCCTAGC GCTGAAAAAC AAGGCTCTGA CGATTACCCC CTGCCCATAA 8442

AATTTGCCTA GTCAAAATAA AAGATGCCGA GTCTATAAAA GCGCAAGGAC AGTTCAGGAG 8502

GTGGCTCGCT CCCTCACCGA CCCTCTGGTC ACGGAGACTC ACCTTGGGGA TCCATCCTCT 8562

CCAAGCGGCC TCGGTTGAGA CGCCTTCCGT GGGACCGTCT CCCGGCCTCG GCACCTCCTG 8622

AACTGCTCCT CCCAAGGTAA GTCTCCTCTC AGGTCGAGCT CGGCTGCCCC TTAGGTAGTC 8682

GCTCCCCGAG GGTCTTTAGA GACACCCGGG TTTCCGCCTG CGCTCGGCTA GACTCTGCCT 8742

TAAACTTCAC TTCCGCGTTC TTGTCTCGTT CTTTCCTCTT CGCCGTCACT GAAAACGAAA 8802

CCTCAACGCC GCCCTCTTGG CAGGCGTCCC GGGGCCAACA TACGCCGTGG AGCGCAGCAA 8862

GGGCTAGGGC TTCCTGAACC TCTCCGGGAG AGGTCTATTG CTATAGGCAG GCCCGCCCTA 8922

GGAGCATTGT CTTCCCGGGG AAGACAAACA 8952

GGA AAA AAA CTC CTT AGG GGG GGA GAT CTA ATC TCC CCC CAT CCC GAT 9000 Gly Lys Lys Leu Leu Lys Gly Gly Asp Leu He Ser Pro His Pro Asp

5 10 15

CAA GAC ATC TCG ATA CTC CCA CTC ATC CCC CTG CGG CAG CAA CAG CAA 9048 Gin Asp He Ser He Leu Pro Leu He Pro Leu Arg Gin Gin Gin Gin 20 25 30

CCA ATT CTA GGG GTC CGG ATC TCC GTT ATG GGA CAA ACA CCT CAG CCT 9096 Pro He Leu Gly Val Arg He Ser Val Met Gly Gin Thr Pro Gin Pro 35 40 45

ACC CAA GCG CTA CTT GAC ACA GGA GCC GAC CTT ACG GTT ATA CCC CAG 9144 Thr Gin Ala Leu Leu Asp Thr Gly Ala Asp Leu Thr Val He Pro Gin 50 55 60

ACA CTC GTG CCC GGG CCG GTA AAG CTC CAC GAC ACC CTG ATC CTA GGC 9192

SUBSTITUTE SHEET Thr Leu Val Pro Gly Pro Val Lys Leu His Asp Thr Leu He Leu Gly 65 70 75 80

GCC AGT GGG CAA ACC AAC ACC CAG TTC AAA CTC CTC CAA ACC CCC CTA 9240 Ala Ser Gly Gin Thr Asn Thr Gin Phe Lys Leu Leu Gin Thr Pro Leu 85 90 95

CAC ATA TTC TTG CCC TTC CGA AGG TCC CCC GTT ATC CTT TCC TCC TGC 9282 His He Phe Leu Pro Phe Arg Arg Ser Pro Val He Leu Ser Ser Cys 100 105 110

CTC TTA GAC ACC CAC AAC AAA TGG ACC ATC ATT GGA AGG GAC GCC CTA 9336 Leu Leu Asp Thr His Asn Lys Trp Thr He He Gly Arg Asp Ala Leu 115 120 125

CAA CAA TGC CAG GGG CTT CTA TAC CTC CCA GAC GAC CCC AGC CCC CAC 9384 Gin Gin Cys Gin Gly Leu Leu Tyr Leu Pro Asp Asp Pro Ser Pro His 130 135 140

CAA TTG CTG CCA ATA GCC ACT CCA AAC ACC ATA GGC CTC GAA CAC CTT 9432 Gin Leu Leu Pro He Ala Thr Pro Asn Thr He Gly Leu Glu His Leu 145 150 155

CCC CCA CCT CCC CAA GTG GAC CAA TTT CCT TTA AAC CTG AGC GCC TCC 9480 Pro Pro Pro Pro Gin Val Asp Gin Phe Pro Leu Asn Leu Ser Ala Ser 160 165 170

AGG CCT 9486

Arg Pro

(2 ) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: (i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 505

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(E) NAME: SEQ ID NO. : 2

(ii) SEQUENCE DESCRIPTION FOR SEQUENCE ID NUMBER: 2

TAC TAC TCG CCT 12 Tyr Tyr Ser Pro

TCC TAC AAT GAC CCT TGC TCG CTA CAA TGC CCC TAC TTG GGC TGC CAA 60

Ser Tyr Asn Asp Pro Cys Ser Leu Gin Cys Pro Tyr Leu Gly Cys Gin

5 10 15 20

GCA TGG ACA TCC GCA TAC ACG GGC CCC GTC TCC AGT CCA TCC TGG AAG 108 Ala Trp Thr Ser Ala Tyr Thr Gly Pro Val Ser Ser Pro Ser Trp Lys 25 30 35

TTT CAT TCA GAT GTA AAT TTC ACC CAG GAA GTC AGC CAA GTG TCC CTT 156 Phe His Ser Asp Val Asn Phe Thr Gin Glu Val Ser Gin Val Ser Leu 40 45 50

CGA CTA CAC TTC TCT AAG TGC GGC TCC TCC ATG ACC CTC CTA GTA GAT 204 Arg Leu His Phe Ser Lys Cys Gly Ser Ser Met Thr Leu Leu Val Asp 55 60 65

GCC CCT GGA TAT GAT CCT TTA TGG TTC ATC ACC TCA GAA CCC ACT CAG 252 Ala Pro Gly Tyr Asp Pro Leu Trp Phe He Thr Ser Glu Pro Thr Gin 70 75 80

CCT CCA CCA ACT TCT CCC CCA TTG GTC CAT GAC TCC GAC CTT GAA CAT 300

Pro Pro Pro Thr Ser Pro Pro Leu Val His Asp Ser Asp Leu Glu His

85 90 95 100

GTC CTA ACC CCC TCC ACG TCC TGG ACG ACC AAA ATA CTC AAA TTT ATC 348 Val Leu Thr Pro Ser Thr Ser Trp Thr Thr Lys He Leu Lys Phe He 105 110 115

CAG CTG ACC TTA CAG AGC ACC AAT TAC TCC TGC ATG GTT TGC GTG GAT 396 Gin Leu Thr Leu Gin Ser Thr Asn Tyr Ser Cys Met Val Cys Val Asp 120 125 130

AGA TCC AGC CTC TCA TCC TGG CAT GTA CTC TAC ACC CCC AAC ATC TCC 444 Arg Ser Ser Leu Ser Ser Trp His Val Leu Tyr Thr Pro Asn He Ser 135 140 145

ATT CCC CAA CAA ACC TCC TCC CGA ACC ATC CTC TTT CCT TCC CTT GCC 492 He Pro Gin Gin Thr.Ser Ser Art Thr He Leu Phe Pro Ser Leu Ala 150 155 160

CTG CCC GCT CCT C 505 Leu Pro Ala Pro 165

S (2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 106

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(E) NAME: SEQ ID NO.: 3

(ii) SEQUENCE DESCRIPTION FOR SEQUENCE ID NUMBER: 3

CC ATC TGG TTC CTT AAT ACC GAA CCC AGC CAA CTG CCT CCC ACC GCC 48 Pro He Trp Phe Leu Asn Thr Glu Pro Ser Gin Leu Pro Pro Thr Ala

5 10 15

CCT CCT CTA CTC CCC CAC TCT AAC CTA GAC CAC ATC CTC GAG CCC TCT 96 Pro Pro Leu Leu Pro His Ser Asn Leu Asp His He Leu Glu Pro Ser 20 25 30

ATA CCA TGG AA 106 He Pro Trp Lys 35

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 4

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(E) NAME: SEQ ID NO.: 4

(ii) SEQUENCE DESCRIPTION FOR SEQUENCE ID NUMBER: 4

GCGGAATTCG TATGATCCTT TATGGTTC 28

SUBSTITUTE SHEET (2 ) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 5

(i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 30

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(E) NAME: SEQ ID NO.: 5

(ii) SEQUENCE DESCRIPTION FOR SEQUENCE ID NUMBER: 5

GCGGCATGCC TAGGTCAGCT GGATAAATTT 30

SUBST

Claims

Claims 1. A method for detecting in a biological sample an antibody to a virion of a human T-cell leukemia virus (HTLV) or to a cell infected with said HTLV, said method comprising providing an antigen encoded by HTLV-type II (HTLV-II) ; contacting a biological sample with said antigen, and detecting formation of an antigen-antibody complex by immunoassay.

2. A test kit for detecting in a biological sample an antibody to a virion of a human T-cell leukemia virus (HTLV) or a cell infected with said HTLV, said kit being compartmentalized to receive in close confinement therein one or more containers which comprise a first container containing an antigen encoded by HTLV type II (HTLV-II) , and a second container containing a means for detecting the formation of an immunocomplex between said antibody and said antigen.

3. The method of claim 1, wherein said antigen comprises the HTLV-II envelope protein or an antigenic segment or analog thereof.

4. The method of claim 1 or 3, wherein said human T-cell leukemia virus is human T-cell leukemia virus type II.

5. The method of claim 4, wherein said HTLV-II- encoded antigen comprises the carboxy-terminal half of the HTLV-II envelope protein.

SUBSTITUTESHEET 6. The method of claim 5, wherein said HTLV-II- encoded antigen comprises HTLV-II RP-IIB or a segment or analog thereof which is reactive with RP-IIB antibodies.

7. The method of claim 6, wherein said HTLV-II- encoded antigen comprises HTLV-II RP-B2 oar a segment or analog thereof which is reactive with RP-B2 antibodies.

8. The method of claim 1, wherein said human T- cell leukemia virus is human T-cell leukemia virus type I and said antigen is reactive with antibodies to HTLV-I.

9. The method of claim 1 or 3, wherein said method further comprises contacting said biological sample with at least one antigen encoded by HTLV-type I.

10. The method of claim 8, wherein said antigen encoded by HTLV-I is an HTLV-I envelope segment or analog reactive with HTLV-I sera and not with HTLV-II sera.

11. The method of claim 10, wherein said antigen encoded by HTLV-I is RP-Bl or a segment or analog thereof which is reactive with RP-Bl antibodies.

12. The method of claim 8, wherein said antigen encoded by HTLV-II and said antigen encoded by HTLV-I are combined and contacted with said biological sample simultaneously.

13. The method of claim 8, wherein said antigen encoded by HTLV-II and antigen encoded by HTLV-I are maintained in separate containers and are contacted with the sample sequentially or with different aliquots of the sample. 14. The method of claim 1 or 3, wherein said immunoassay is a Western blot.

15. The method of claim 1 or 3, wherein said immunoassay is an ELISA.

16. A method for detecting in a biological sample an antibody to a virion of a human T-cell leukemia virus type I (HTLV-I) or to a cell infected with said HTLV-I, said method comprising providing HTLV-I recombinant protein RP-Bl or an antigenic segment or analog thereof; contacting said biological sample with said RP-Bl or said antigenic segment or analog, and detecting formation of an antigen-antibody complex by immunoassay.

17. A test kit for detecting in a biological sample an antibody to human T-cell leukemia virus type I (HTLV-I) , said kit being compartmentalized to receive in close confinement therein one or more containers which comprise a first container containing HTLV-I recombinant protein RP-Bl or an antigenic segment thereof, and a second container containing a means for detecting the formation of an immunocomplex between said antibody and said antigen.

18. The method of claim 16, wherein said immunoassay is a Western blot.

19. The method of claim 16, wherein said immunoassay is an ELISA.

BSTITUTESHEET 20. A peptide which is the RP-IIB peptide or a segment or analog, thereof, which is reactive with RP- IIB antibodies.

21. The peptide of claim 20, wherein said peptide is RP-B2 or a segment or analog, thereof, which is reactive with RP-B2 antibodies.

22. The peptide of claim 20, wherein said peptide is not reactive with antibodies specific for RP-Bl.

23. Purified nucleic acid encoding the RB-IIB peptide or a segment or analog, thereof, which is reactive with RP-IIB antibodies.

24. Purified nucleic acid encoding the RP-B2 peptide or a segment or analog, thereof, which is reactive with RP-B2 antibodies.

SUBSTITUTE SHEET