WO1995032290A2 - Non-a/non-b/non-c/non-d/non-e hepatitis agents and molecular cloning thereof - Google Patents

Non-a/non-b/non-c/non-d/non-e hepatitis agents and molecular cloning thereof Download PDF

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WO1995032290A2
WO1995032290A2 PCT/US1995/005980 US9505980W WO9532290A2 WO 1995032290 A2 WO1995032290 A2 WO 1995032290A2 US 9505980 W US9505980 W US 9505980W WO 9532290 A2 WO9532290 A2 WO 9532290A2
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dna
seq
hepatitis
abcde
clone
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PCT/US1995/005980
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French (fr)
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WO1995032290A3 (en
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Robert H. Purcell
Jungsuh P. Kim
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Genelabs Technologies, Inc.
THE UNITED STATES OF AMERICA represented by THE SECRETARY OF THE DEPARTMENT OF HEALTH AND HUMAN SERVICES
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Priority to AU25491/95A priority Critical patent/AU2549195A/en
Publication of WO1995032290A2 publication Critical patent/WO1995032290A2/en
Publication of WO1995032290A3 publication Critical patent/WO1995032290A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • This invention relates methods for the isolation of sequences corresponding to N-(ABCDE) hepatitis agents, including immunological and hybridization screening meth ⁇ ods, and further, to DNA and cDNA libraries useful in such methods.
  • Valenzuela, P., et al . Nature 298:344 (1982). Valenzuela, P., et al., in HEPATITIS B. (I. Millman, et al., Eds.) Plenum Press, pages 225-236 (1984).
  • Viral hepatitis resulting from a virus other than hepatitis A virus (HAV) and hepatitis B virus (HBV) has been referred to as non-A, non-B hepatitis (NANBH) .
  • HAV hepatitis A virus
  • HBV hepatitis B virus
  • NANBH enterically transmitted NANBH
  • ET- NANBH hepatitis E virus
  • a second NANB virus type known as parenterally transmitted NANBH, or PT-NANBH
  • PT-NANBH is transmitted by parenteral routes, typically by exposure to blood or blood products.
  • parenteral routes typically by exposure to blood or blood products.
  • approximately 10% of transfusions cause PT-NANBH infection, and about half of these go on to a chronic disease state (Divag) .
  • HCV seroconversion per unit transfused was decreased to less than 1% among heart surgery patients.
  • RNA isolated from infected chimpanzee sera has been used to construct cDNA libraries in an expression vector for immunoscreening with chronic-state human PT- NANBH serum. This procedure identified a PT-NANBH specific cDNA clone and the viral sequence was then used as a probe to identify a set of overlapping fragments making up 7,300 contiguous basepairs of a PT-NANBH viral agent.
  • HCV hepatitis C virus
  • EPO patent application 88310922.5 the sequence of HCV is presented in EPO patent application 88310922.5, filed 11/18/88.
  • the full-length sequence ( ⁇ 9,500 nt) of HCV is now available.
  • Primate transmission studies conducted at the Centers for Disease Control (CDC; Phoenix, AZ, 1973-1975; 1978- 1983) originally provided substantial evidence for the existence of multiple agents of non-A, non-B (NANBH) : the primary agents of NANB are now recognized as being associated with infection by HCV and HEV (see above) .
  • the present invention includes a method of obtaining immunogenic polypeptides associated with non-A, non-B, non-C, non-D, non-E hepatitis agent (N- (ABCDE) ) infection.
  • phage are prepared from a library selected from the group consisting of: MY 620 DNA source; MY 620 cDNA source; MY 670 DNA source; and MY 670 cDNA source. These libraries are deposited at Genelabs Technologies, Incorporated, 505 Penobscot Drive, Redwood City, CA 94063.
  • the phage are plated to form plaques and the phage plaques are screened for production of polypeptides immunoreactive with N-(ABCDE) serum.
  • the serum used for screening the plaques can be from any N-(ABCDE) source, including human, mystax monkey or cynomolgus monkey serum.
  • the invention further includes a method of obtaining non-A, non-B, non-C, non-D, non-E hepatitis agent (N-
  • phage are prepared that have insert sequences from a library selected from the group consisting of: MY 620 DNA source; MY 620 cDNA source; MY 670 DNA source; and MY 670 cDNA source.
  • the phage are plated to form plaques.
  • the plaques are then screened for production of polypeptides immunoreactive with N-(ABCDE) serum. Phage are isolated that produce polypeptides immunoreactive with N-(ABCDE) serum and the insert sequence is used to prepare hybridization probes.
  • Phage are once again prepared from a library selected from the group consisting of MY 620 DNA source, MY 620 cDNA source, MY 670 DNA source, and MY 670 cDNA source, and plated to form plaques.
  • the resulting plaques are screened using the hybridization probes.
  • the hybridization probes can be selected from the sequences obtained by the method of the present invention.
  • the invention also includes the following libraries: MY 620 DNA source; MY 620 cDNA source; MY 670 DNA source; and MY 670 cDNA source.
  • the present invention includes the isolation of polypeptide antigens that are immunoreactive with sera infected with a non-A, non-B, non-C, non-D, non-E hepatitis agent.
  • Exemplary embodiments of the present invention include polypeptide antigens where an immunoreactive portion of the antigen is homologous to a polypeptide encoded by a sequence selected from the group consisting of: SEQ ID NO:11 through SEQ ID NO:16, SEQ ID NO:39 through SEQ ID NO:43, SEQ ID NO:74 through SEQ ID NO:104, and SEQ ID NO:106.
  • SEQ ID NO:11 through SEQ ID NO:16 SEQ ID NO:39 through SEQ ID NO:43
  • SEQ ID NO:74 through SEQ ID NO:104
  • SEQ ID NO:106 a number of other antigenic peptides, useful in the practice of the present invention, are disclosed herein.
  • Selected antigens of the present are encoded by the nucleic acid sequences presented as SEQ ID NO:11 through SEQ ID NO:16, SEQ ID NO:39 through SEQ ID NO:43, SEQ ID NO:74 through SEQ ID NO:104, and SEQ ID NO:106.
  • These antigens may include heterologous protein sequences (i.e., they may be fused polypeptides) , such as sequences encoding / 9-galactosidase or glutathione-S-transferase.
  • the present invention also includes an expression system for expressing an antigenic polypeptide having an immunoreactive portion that is immunoreactive with sera infected with non-A, non-B, non-C, non-D, non-E hepatitis agent.
  • the expression system typically includes a host capable of supporting expression of an open reading frame in a selected expression vector, where the selected expression vector includes an open reading frame of sequences encoding an immunoreactive portion of the polypeptide antigens described above.
  • One useful expression vector is lambda gtll: other useful expression vectors are known in the art.
  • the present invention discloses a method of producing a polypeptide that is immunoreactive with N- (ABCDE) hepatitis sera.
  • the polypeptide is produced by introducing a selected expression vector containing an open reading frame having sequences encoding the polypeptide antigen into a host capable of supporting expression of an open reading frame in the selected expression vector.
  • the host cell is then cultured under conditions resulting in the expression of the open reading frame sequence.
  • One useful expression vector is lambda gtll phage vector and the host is Escherichia coli .
  • a further embodiment of the present invention is a cloning vector capable of expressing under suitable conditions an antigenic polypeptide having an immunoreactive portion that is immunoreactive with sera infected with non-A, non-B, non-C, non-D, non-E hepatitis agent.
  • the antigen is homologous to one of the polypeptides identified by the methods of the present invention and includes those polypeptides encoded by a sequence selected from the group consisting of: SEQ ID NO:11 through SEQ ID NO:16, SEQ ID NO:39 through SEQ ID NO:43, SEQ ID NO:74 through SEQ ID NO:104, and SEQ ID NO:106.
  • One embodiment of the present invention includes a recombinantly produced N-(ABCDE) hepatitis agent poly ⁇ nucleotide that encodes a polypeptide which is immuno ⁇ reactive with N-(ABCDE) hepatitis infected sera, where said polynucleotide is selected from the group consisting of: SEQ ID NO:11 through SEQ ID NO:16, SEQ ID NO:39 through SEQ ID NO:43, SEQ ID NO:74 through SEQ ID NO:104, and SEQ ID NO:106.
  • the polynucleotide can correspond to other sequences obtained by the method of the present invention.
  • Another embodiment of the present invention includes a recombinantly produced N-(ABCDE) hepatitis agent polypeptide which is homologous to a polypeptide encoded by a sequence selected from the group consisting of: SEQ ID NO:11 through SEQ ID NO:16, SEQ ID NO:39 through SEQ ID NO:43, SEQ ID NO:74 through SEQ ID NO:104, and SEQ ID NO:106.
  • the recombinant polypeptide can be homologous to other polypeptides antigens obtained by the method of the present invention.
  • a diagnostic kit for use in screening serum containing antibodies specific against N-(ABCDE) hepatitis infection is also included in the invention.
  • the kit includes a recombinant N-(ABCDE) hepatitis polypeptide antigen.
  • polypeptide antigens are polypeptides where an immunoreactive portion of said antigen is homologous to a polypeptide encoded by a sequence selected from the group consisting of: SEQ ID NO:11 through SEQ ID NO:16, SEQ ID NO:39 through SEQ ID NO:43, SEQ ID NO:74 through SEQ ID NO:104, and SEQ ID NO:106.
  • the kit also includes means for detecting the binding of said antibodies to the antigen.
  • One means for detecting the binding of antibodies to the antigen includes a solid support to which the N-(ABCDE) hepatitis polypeptide antigen is attached and a reporter-labelled anti-human antibody, where binding of serum antibodies to the antigen can be detected by binding of the reporter-labelled anti- body to said serum antibodies.
  • The can be used in a method of detecting N-(ABCDE) hepatitis agent infection in a primate. In this method the serum from a N-(ABCDE) hepatitis test primate is reacted with a recombinant N- (ABCDE) hepatitis polypeptide antigen and the antigen is examined for the presence of bound antibody.
  • the present invention also includes a diagnostic kit for use in screening samples containing N-(ABCDE) hepatitis agent nucleic acids.
  • This kit contains primers having sequences specific to N-(ABCDE) hepatitis agents. For example, such sequences can be selected from the group consisting of: SEQ ID NO:11 through SEQ ID NO:16, SEQ ID NO:39 through SEQ ID NO:43, SEQ ID NO:74 through SEQ ID NO:104, and SEQ ID NO:106.
  • the primer may contain reporter moieties for hybridization detection.
  • These primers may be used in a method of detecting N-(ABCDE) hepatitis agent nucleic acid in a primate. In the method a nucleic acid sample is obtained from the primate subject.
  • the sample is combined with at least one primer containing sequences specific to N-(ABCDE) hepatitis agents.
  • the presence of N-(ABCDE) hepatitis agent nucleic acid/primer complexes, formed by hybridization of the N- (ABCDE) hepatitis nucleic acid with primer, is then detected.
  • sets of primers can be used to identify target N-(ABCDE) hepatitis agent nucleic acid in a sample by use of polymerase chain reaction amplification.
  • a capture moiety may be included in a primer to allow capture of target sequences to which it binds (e.g., biotin incorporated, avidin capture) .
  • Figure 2 the GB sera use in cross challenge to mystax infected with passaged sera derived from PNF 2161 is diagrammed.
  • Figure 3 illustrates the results of a Western blot analysis of the membrane containing crude lysates containing the pGEX-GLI-D6 and pGEX-GLI-D19 encoded antigens of the present invention.
  • Figures 4A and 4B illustrate the results of Western blots of the partially purified Dl, D6 and D19 fusion proteins using mystax (My 88) serum ( Figure 4A) and N-
  • FIGS. 5A, 5B and 5C illustrate the result of
  • FIG. 5A is a photograph of a coomaisse blue stained polyacrylamide gel on which the samples were separated.
  • Figure 5B shows a Western blot analysis of the
  • Figure 5A polyacrylamide gel transferred to a membrane and probed with JFA serum.
  • Figure 5B the arrows indicate the locations of the SJ26-17A and SJ26-1A fusion proteins, first and second panels, respectively.
  • Figure 5C shows the Western blot analysis of Figure 5A polyacrylamide gel transferred to a membrane and probed with normal human serum.
  • Figure 6 schematically illustrates the overlap between clone 17A and WT54.
  • Figure 7 schematically represents the steps taken in the polymerase chain reaction linking experiments for clones JFA-17A and WT54. Detailed Description of the Invention
  • nonA/nonB/nonC/nonD/nonE hepatitis viral agent N-(ABCDE) means a virus, virus type, or virus class which (i) is transmissible in primates (e . g. , mystax, cynomolgus and marmoset monkeys, chimpanzees, or humans) , and (ii) is serologically distinct from hepatitis A virus (HAV) , hepatitis B virus (HBV) , hepatitis C virus (HCV) , hepatitis D virus, and hepatitis E (HEV) .
  • HAV hepatitis A virus
  • HBV hepatitis B virus
  • HCV hepatitis C virus
  • HEV hepatitis D virus
  • HEV hepatitis E
  • Two nucleic acid fragments are considered to have "homologous hybridizable" sequences if they are capable of hybridizing to one another (i) under typical hybridization and wash conditions, as described, for example, in Maniatis, et al . , pages 320-328, and 382-389, or (ii) using reduced stringency wash conditions that allow at most about 25-30% basepair mismatches, for example: 2 x SSC, 0.1% SDS, room temperature twice, 30 minutes each; then 2 x SSC, 0.1% SDS, 37°C once, 30 minutes; then 2 x
  • homologous nucleic acid strands contain 15-25% basepair mismatches, even more preferably 5-15% basepair mismatches. These degrees of homology can be selected by using wash conditions of appropriate stringency for identification of clones from gene libraries (or other sources of genetic material) , as is well known in the art.
  • Two amino acid sequences or two nucleotide sequences are considered homologous (as this term is preferably used in this specification) if they have an alignment score of >5 (in standard deviation units) using the program ALIGN with the mutation gap matrix and a gap penalty of 6 or greater (Dayhoff) .
  • the two sequences (or parts thereof, preferably at least 35 amino acids in length) are homologous if their amino acids alignments are greater than or equal to 40%, preferably 60% and more preferably 80% using the ALIGN program mentioned above.
  • a DNA or cDNA fragment is "derived from" N- (ABCDE) viral agents if it has the same or substantially the same basepair sequence as a cloned region of a N- (ABCDE) viral agent genome.
  • a protein is "derived from" N-(ABCDE) viral agents if it is encoded by an open reading frame of a DNA or RNA fragment derived from a N-(ABCDE) viral agent or displays homology as noted under Example 3 above.
  • nucleic acid sequences when referring to sequences which encode a protein, polypeptide, or peptide, is meant to include degenerative nucleic acid sequences which encode homologous .protein, polypeptide or peptide sequences as well as the disclosed sequence.
  • a third amino acid sequence will be internally consistent with the known sequences if each amino acid in the third sequence is identical to at least one of amino acids in the known sequences.
  • NANBH other than HCV, in the chronic-phase plasma of case PNF-2161.
  • Figure 1 illustrates serial passage of non-HCV hepatitis in both species of primate and further illustrates that the etiologic agent is chloroform- resistant.
  • PNF 2161 is the original non-C serum that was used to inoculate two chimpanzees (CH1323, with 1 ml. of pooled sera, and CH1356, with 10 ml. of plasma) , tamarins (Sanguinis mystax; MY, with 1 ml. of plasma) and cynos (Cynomolgus macaques; CY, with 1 ml. of plasma) . These animals were then monitored for elevated ALT or SICD values (Bradley, et al . , 1987, op.
  • MY 131 had very significant elevated ALT values.
  • the liver of MY131 was obtained at or near the peak of ALT activity.
  • a liver homogenate was prepared and diluted.
  • Ten percent and 1% liver solutions were inoculated into S . mystax primates.
  • a 1% liver solution was inoculated into cynos primates.
  • For each inoculation the liver was either treated or not treated with chloroform prior to injection.
  • ALT and SICD values were determined for each animal.
  • Tamarins MY 88 and MY 190 also developed non-C hepatitis.
  • N-(ABCDE) sera have also been identified including SCH (Example 1) .
  • Animal transmission and cross-challenge studies of this type indicate the basic methodology used in the identification of new blood-born pathogenic viruses.
  • cDNA and DNA libraries were prepared from infected sera in the expression vector lambda gtll (Examples 2, 3 and 16) . cDNA and DNA sequences were then selected for the expression of peptides which are immunoreactive with N-(ABCDE) hepatitis infected sera.
  • First round screening was typically performed using the same sera that was used to generate the phage library, or, alternatively, a closely related sera. It is also possible to screen with other suspected N-(ABCDE) sera.
  • Recombinant proteins identified by this approach provide candidates for peptides which can serve as substrates in diagnostic tests. Further, the nucleic acid coding sequences identified by this approach serve as useful hybridization probes for the identification of further N-(ABCDE) hepatitis coding sequences.
  • Example 2 The sera described above were used to generate cDNA and DNA libraries in lambda gtll (Examples 2, 3 and 16). In the method illustrated in Example 2, infected serum was precipitated in 8% PEG without dilution, and the libraries were generated from the resulting pelleted virus. Sera from infected human sources were treated in the same fashion.
  • ultracentrifugation can be used to pellet particulate agents from infected sera or other biological specimens.
  • serum ranging up to 2 ml
  • serum was diluted to approximately 10 ml with PBS and was centrifuged for a minimum of 2 hours at 40,000 rpm (approximately 110,000 x g) in the Ti70.1 rotor (Beckman Instruments, Fullerton, CA) at 4°C.
  • the supernatant was the aspirated and the pellet extracted by standard nucleic acid extraction techniques.
  • cDNA libraries were generated using random primers in reverse transcription reactions with RNA extracted from pelleted sera as starting material.
  • DNA libraries were generated by proteinase K treatment and SDS lysis of pelleted sera, followed by the addition of Klenow fragment of DNA polymerase and random primers to the nucleic acid.
  • the resulting molecules, cDNA or DNA were ligated to SISPA (Reyes, et al . , (1991)) linker primers and expanded in a non-selective manner, and then cloned into a suitable vector, for example, lambda gtll, for expression and screening of peptide antigens, and the lambda gtlO vector, for hybridization screening.
  • SISPA Reyes, et al . , (1991)
  • Lambda gtll is a particularly useful expression vector which contains a unique EcoRI insertion site 53 base pairs upstream of the translation termination codon of the ?-galactosidase gene.
  • an inserted sequence is expressed as a ?-galactosidase fusion protein which contains the N-terminal portion of the ?-galactosidase gene product, the heterologous peptide, and optionally the C-terminal region of the ?-galactosidase peptide (the C— terminal portion being expressed when the heterologous peptide coding sequence does not contain a translation termination codon) .
  • This vector also produces a temperature-sensitive repressor (cI857) which causes viral lysogeny at permissive temperatures, e.g., 32°C, and leads to viral lysis at elevated temperatures, e.g., 42°C.
  • Advantages of this vector include: (1) highly efficient recombinant clone generation, (2) ability to select lysogenized host cells on the basis of host-cell growth at permissive, but not non-permissive, temperatures, and (3) high levels of recombinant fusion protein production. Further, since phage containing a heterologous insert produces an inactive ?-galactosidase enzyme, phage with inserts are typically identified using a yff-galactosidase colored-substrate reaction.
  • Examples 2, 3 and 16 describe the preparation of a cDNA and DNA library for each of the following N-(ABCDE) hepatitis sera: MY 131, MY 190, MY 620, MY 670, PNF 2161, JFA, SCH and DEN. These libraries were immunoscreened using N-(ABCDE) hepatitis positive human or mystax sera (Examples 4, and 7-11). A number of lambda gtll clones were identified which were immunoreactive with at least one of the sera. Immunopositive clones were plaque-purified and their immunoreactivity retested. Also, the immunoreactivity of the clones with pre-inoculu mystax and/or normal human sera was also tested.
  • the clone inserts were isolated by EcoRI digestion following polymerase chain reaction amplification. The inserts were purified then radiolabelled and used as hybridization probes against membrane bound normal human DNA, normal mystax DNA and bacterial DNA (control DNAs) (Example 6) .
  • clones that were (i) immunoreactive with the N-(ABCDE) hepatitis test sera, (ii) exogenous to human, normal, and bacterial genomes, (iii) not immunologically reactive with pre-immune mystax and/or normal human sera, and (iv) had unique nucleic acid sequences when compared with one another.
  • the latter may indicate the isolation of multiple viruses or the isolation of different immunogenic regions from the same genome.
  • sequences of these clones are presented in the Sequence Listing.
  • the sequences of the cloned inserts when searched against the "GENBANK” sequence library, were not found to have significant homology to any known sequences, including those from known hepatitis virus sequences. Other characteristics of a number of the sequenced clones follow here.
  • the D19 clone (MY 190 DNA source library) also was shown to be exogenous to normal human, tamarin and bacterial DNA.
  • the clone has a large open reading frame (104 bp) , in frame with the /3-galactosidase gene of the lambda gtll vector (SEQ ID NO:17).
  • the antigen encoded by D19 was shown to be immunoreactive with 2/9 infected mystax sera.
  • Clone 17A (JFA DNA source) was shown to be exogenous to normal human and bacterial DNA.
  • the clone has a large open reading frame (590 bp) , in frame with the ⁇ - galactosidase gene of the lambda gtll vector (SEQ ID NO:36).
  • the 17A antigen has been expressed as a GST fusion protein and tested immunopositive with JFA serum.
  • Clone IA JFA DNA source
  • the clone has a large open reading frame (467 bp) , in frame with the ⁇ - galactosidase gene of the lambda gtll vector (SEQ ID NO:37).
  • the antigen encoded by clone IA has been expressed as a GST fusion protein and tested immunopositive with JFA serum and negative with normal human sera by Western blot analysis.
  • Some clones have multiple insert sequences as indicated by internal SISPA primers (Example 2) : for example, JFA clone 4B11 (SEQ ID NO:38), D12-3 (SEQ ID N0:46), D31-2 (SEQ ID NO:48) and D76 (SEQ ID NO:50) have 3 inserts; and R27 has four inserts (SEQ ID NO:33).
  • the insert can be (i) fractionated into discrete sequences by restriction enzyme digestion with EcoRI or NotI , or (ii) portions of the insert separately PCR amplified by sequence specific primers.
  • Each resulting individual region of the cloned sequence can be subcloned into, for example, lambda gtll or pGEX-GLI and immunoscreened as described above. This allows identification of specific regions responsible for the immunoreactivity (see Epitope Mapping, below) .
  • Clone 470-20-1 (PNF2161 cDNA source) was isolated by immunoscreening with the same cloning source. The clone was not reactive with normal human sera. The clone has a large open reading frame (203 base pairs; SEQ ID NO:106), in-frame with the ,9-galactosidase gene of the lamdba gtll vector. The clone is exogenous by genomic DNA hybridization analysis and genomic PCR analysis, using human, yeast and E. coli genomic DNAs. The sequence was present in PNF2161 serum as determined by RT-PCR. The sequence was also detected in sucrose density gradient fractions at densities consistent with the sequence banding in association with a virus-like particle. Further sequences (PNF2161-470-20-1 EXT1; SEQ ID NO:106), in-frame with the ,9-galactosidase gene of the lamdba gtll vector. The clone is exogenous by genomic DNA hybridization analysis and genomic PCR
  • N-(ABCDE) hepatitis antigens can be obtained from the libraries of the present invention using the screening methods described above.
  • the libraries described above have been deposited with the American Type Culture Collection, 12301 Parklawn Dr., Rockville MD 20852 and have been assigned the following designations: MY 131 cDNA source, ATCC 75273; MY 131 DNA source, ATCC 75270; MY 190 DNA source, ATCC 75284; MY 190 cDNA source; JFA cDNA source, ATCC 75272; JFA DNA source, ATCC 75271; SCH cDNA source, ATCC 75283; SCH DNA source, ATCC 75282; PNF 2161 CDNA source, ATCC 75268; PNF 2161 DNA source, ATCC 75269; DEN cDNA source, ATCC 75417; and DEN DNA source, ATCC 75418.
  • the MY 620 cDNA source, MY 620 DNA source, MY 670 cDNA source, MY 670 DNA source and subtracted SCH DNA source libraries are deposited at Genelabs Technologies, Incorporated, 505 Penobscot Drive, Redwood City, CA 94063.
  • the antigen-encoding DNA fragment can be subcloned.
  • the subcloned insert can then be fragmented by partial DNase I digestion to generate random fragments or by specific restriction endonuclease digestion to produce specific subfragments.
  • the resulting DNA fragments can be inserted into the lambda. gtll vector and subjected to immunoscreening in order to provide an epitope map of the cloned insert.
  • the DNA fragments can be employed as probes in hybridization experiments to identify overlapping N-(ABCDE) hepatitis sequences, and these in turn can be further used as probes to identify a set of contiguous clones.
  • the generation of sets of contiguous clones allows the elucidation of the sequence of a given N-(ABCDE) hepatitis agent's genome.
  • the following illustrates several approaches for the use of cloned insert-derived DNA to identify clones carrying other N-(ABCDE) hepatitis sequences.
  • the insert of clone JFA-17A was isolated and used as a hybridization probe against the individual cDNA or DNA libraries established in a lambda vector (Example 10, Sections 10D and 10E) .
  • a lambda vector Example 10, Sections 10D and 10E
  • the insert of the overlapping clone can be isolated by restriction enzyme digestion of the clone WT54, followed by electrophoretic fractionation and electroelu- tion.
  • the isolated insert is then treated with DNase I to generate random fragments and the resulting digested frag ⁇ ments are inserted into lambda gtll phage vectors for immunoscreening to yield other immunoreactive regions.
  • any of the above-described clone inserts can be used in a similar manner to probe the cDNA and DNA libraries generated in a vector, such as lambda gtlO or "LAMBDA ZAP II". Entire inserts of specific subfragments of any clone may be isolated by polymerase chain reaction or after cleavage with restriction endonucleases. These fragments can be used as radiolabelled probes against any selected library. In particular, the 5' and 3' terminal sequences of the clone inserts are useful as probes to identify additional clones.
  • the clone inserts can be used to screen other libraries, for example, the D19 clone insert can be used to screen libraries generated from other human N-(ABCDE) hepatitis sera (such as SCH source libraries) .
  • sequences provided by the 5' end of cloned inserts are useful as sequence specific primers in first-strand cDNA or DNA synthesis reactions (Maniatis et al . ; Scharf et al . ) .
  • specifically primed MY 190 cDNA and DNA libraries can be prepared by using a D19 specific primer on My 190 nucleic acids as a template.
  • the second-strand of the new cDNA is synthesized using RNase H and DNA polymerase I. The above procedures identify or produce DNA/cDNA molecules corresponding to nucleic acid regions that are 5' adjacent to the known clone insert sequences.
  • N-(ABCDE) hepatitis agent can be used to identify further flanking se- quences, and so on, to identify the sequences composing the entire genome for a given N-(ABCDE) hepatitis agent.
  • the polynucleotides can be cloned and immunoscreened to identify specific sequences encoding N-(ABCDE) hepatitis antigens.
  • extension clones containing further sequences of interest, have been obtained for clone PNF 470-20-1 (SEQ ID NO:106): for example, extension clone (PNF2161- 470-20-1 EXT1, SEQ ID NO:104; Example 7).
  • the recombinant peptides of the present invention can be purified by standard protein purification procedures which may include differential precipitation, molecular sieve chromatography, ion-exchange chromatography, isoelectric focusing, gel electrophoresis and affinity chromatography.
  • a fusion protein such as the / 9-galactosidase fusion proteins described above
  • the fused protein can be isolated readily by affinity chromatography, by passing cell lysis material over a solid support having surface-bound anti-yff-galactosidase antibody.
  • affinity chromatography purification of a yff-galactosi- dase/fusion protein, derived from D19 coding sequences, by affinity chromatography is described in Example 12.
  • Fusion proteins containing the polypeptide antigens of the present invention fused with the glutathione-S- transferase (Sj26) protein have also been expressed using the pGEX-GLI vector system in E. coli JM101 cells (Examples 9D, 9E, and 12) .
  • the fused Sj26 protein can be isolated readily by glutathione substrate affinity chromatography (Smith, et al . ) .
  • Expression and partial purification of the D6, and D19 as GST (sj26) fusion proteins is described in Examples 9C, 9D and 12, and is applicable to any of the other soluble, induced antigens coded by sequences described by the present invention.
  • Insoluble GST (sj26) fusion proteins (such as, the GST fusions containing the JFA-1A and JFA-17A antigens) have been purified by preparative gel electrophoresis.
  • an expression vector such as the lambda gtll or pGEX vectors described above, containing N-(ABCDE) antigen coding sequences and expression control elements which allow expression of the coding regions in a suitable host.
  • the control elements generally include a promoter, translation initiation codon, and translation and transcription termination se ⁇ quences, and an insertion site for introducing the insert into the vector.
  • the DNA encoding the desired antigenic polypeptide can be cloned into any number of commercially available vectors to generate expression of the polypeptide in the appropriate host system.
  • These systems include: baculovirus expression (Reilly, et al . ; Beames, et al . ; Pharmigen; Clontech) , expression in bacteria (Ausubel, et al . ; Clontech), expression in yeast (Goeddel; Guthrie and Fink) , expression in mammalian cells (Clontech; Gibco- BRL) .
  • baculovirus expression Reilly, et al . ; Beames, et al . ; Pharmigen; Clontech
  • yeast Goeddel; Guthrie and Fink
  • mammalian cells Clontech; Gibco- BRL
  • N-(ABCDE) hepatitis polypeptide antigens are typically isolated from lysed cells or culture media. Purification can be carried out by methods known in the art including salt fractionation, ion exchange chromatography, and affinity chromatography. Immunoaffinity chromatography can be employed using antibodies generated based on the N-(ABCDE) hepatitis antigens identified by the methods of the present invention.
  • N-(ABCDE) hepatitis polypeptide antigens may also be isolated from N-(ABCDE) hepatitis agent particles (see below) .
  • Antigenic regions of polypeptides are generally relatively small, typically 7 to 10 amino acids in length. Smaller fragments have been identified as antigenic regions. N-(ABCDE) hepatitis polypeptide antigens are identified as described above. The resulting DNA coding regions can be expressed recombinantly either as fusion proteins or isolated polypeptides. In addition, some amino acid sequences can be conveniently chemically synthesized (Applied Biosystems, Foster City CA) . Antigens obtained by any of these methods may be directly used for the generation of antibodies or they may be coupled to appropriate carrier molecules. Many such carriers are known in the art and are commercially available (e . g. , Pierce, Rockford IL) .
  • the invention includes specific antibodies directed against the polypeptide antigens of the present invention.
  • a host animal such as a rabbit
  • the purified antigen or fused protein antigen may be generated using a variety of coding sequence derived from other proteins, such as ⁇ -galac- tosidase or glutathione-S-transferase.
  • the host serum or plasma is collected following an appropriate time inter ⁇ val, and this serum is tested for antibodies specific against the antigen.
  • Example 13 describes the production of rabbit serum antibodies which are specific against the D19 antigens in the SJ26/D19 hybrid protein. These techniques are equally applicable to the other antigens of the present invention.
  • the gamma globulin fraction or the IgG antibodies of immunized animals can be obtained, for example, by use of saturated ammonium sulfate precipitation or DEAE Sephadex chromatography, or other techniques known to those skilled in the art for producing polyclonal antibodies.
  • purified antigen or fused antigen pro ⁇ tein may be used for producing monoclonal antibodies.
  • the spleen or lymphocytes from an immunized animal are removed and immortalized or used to prepare hybridomas by methods known to those skilled in the art.
  • a human lymphocyte donor is selected.
  • a donor known to be infected with a N-(ABCDE) hepatitis agent may serve as a suitable lymphocyte donor.
  • Lymphocytes can be isolated from a peripheral blood sample.
  • Epstein-Barr virus (EBV) can be used to immortalize human lymphocytes or a human fusion partner can be used to produce human-human hybridomas.
  • Primary in vitro sensitization with viral specific polypeptides can also be used in the generation of human monoclonal antibodies.
  • Antibodies secreted by the immortalized cells are screened to determine the clones that secrete antibodies of the desired specificity, for example, by using the ELISA or Western blot method (Ausubel et al . ) .
  • N-(ABCDE) antigens When N-(ABCDE) antigens are identified, typically through plaque immunoscreening as described above, the antigens can be expressed and purified. The antigens can then be screened rapidly against a large number of suspected N-(ABCDE) hepatitis sera using alternative immunoassays, such as, ELISAs or Protein Blot Assays (i . e . , Westerns) employing the isolated antigen peptide.
  • the antigen polypeptides fusion can be isolated as described above, usually by affinity chromatography to the fusion partner such as jS-galactosidase or glutathione-S- transferase.
  • the antigen itself can be purified using antibodies generated against it (see below) .
  • a general ELISA assay format is presented in the Materials and Methods section below. Harlow, et al . , describe a number of useful techniques for immunoassays and antibody/antigen screening.
  • the purified antigen polypeptide or fusion polypeptide containing the antigen of interest is attached to a solid support, for example, a multiwell polystyrene plate.
  • Sera to be tested are diluted and added to the wells. After a period of time sufficient for the binding of antibodies to the bound antigens, the sera are washed out of the wells.
  • a labelled reporter antibody is added to each well along with an appropriate substrate: wells containing antibodies bound to the purified antigen polypeptide or fusion polypeptide containing the antigen are detected by a positive signal.
  • Example 15 A typical format for protein blot analysis using the polypeptide antigens of the present invention is presented in Example 15. General protein blotting methods are described by Ausubel, et al . In Example 15, fusion proteins containing the antigens JFA-1A, JFA-17A, D6 and D19 were used to screen a number of sera samples. The results presented in Example 15 demonstrate that several different source N(ABCDE) Hepatitis sera are immunoreactive with these polypeptide antigens.
  • polypeptide antigens of the present invention can, by these methods, be rapidly screened against panels of N-(ABCDE) hepatitis serum samples.
  • N-(ABCDE) hepatitis agents may be propagated in the animal model systems described above.
  • the N-(ABCDE) hepatitis agents described in the present specification have the advantage of being capable of infecting marmoset and cynos monkeys. This provides a convenient and accessible animal model as well as an animal model that discriminates against the propagation of HCV.
  • primary hepatocytes obtained from infected animals can be cultured in vitro .
  • a serum-free medium supplemented with growth factors and hormones, has been described which permits the long-term maintenance of differentiated primate hepatocytes (Lanford, et al . ; Jacob, et al . , 1989, 1990, 1991).
  • immortalized cultures of infected cells may also be generated. For example, primary liver cultures may be fused to a variety of cells (like HepG2) to provide stable immortalized cell lines.
  • Primary hepatocyte cell cultures may also be immortalized by introduction of oncogenes or genes causing a transformed phenotype.
  • oncogenes or genes can be derived from a number of sources known in the art including SV40, human cellular oncogenes and Epstein Barr Virus.
  • the un-infected primary hepatocytes may be infected by exposing the cells in culture to the N-(ABCDE) hepatitis agents either as partially purified particle preparations (prepared, for example, from infected sera by differential centrifugation and/or molecular sieving) or in infectious sera. These infected cells can then be propagated and the virus passaged by methods known in the art.
  • regions of the N-(ABCDE) hepatitis agent genomic information can be introduced by recombinant means into the hepatocyte cells. Such recombinant manipulations allow the individual expression of individual components of the N-(ABCDE) hepatitis agent genomes.
  • RNA samples can be prepared from infected tissue or, in particular, from infected cell cultures. The RNA samples can be fractionated on gels and transferred to membranes for hybridization analysis using probes derived from the cloned N-(ABCDE) hepatitis sequences.
  • N-(ABCDE) hepatitis particles may be isolated from infected sera, infected tissue, the above-described cell culture media, or the cultured infected cells by methods known in the art. Such methods include techniques based on size fractionation (i.e., density centrifugation, precipitation, ultracentrifugation) , using anionic and/or cationic exchange materials, separation on the basis of hydrophilic properties, and affinity chromatography. During the isolation procedure the N-(ABCDE) hepatitis agents can be identified using the anti-N-(ABCDE) hepatitis antibodies of the present invention or by using hybridization probes based on identified N-(ABCDE) hepatitis agent nucleic acid sequences.
  • Antibodies directed against the N-(ABCDE) hepatitis agents can be used in purification of N-(ABCDE) hepatitis particles through immunoaffinity chromatography (Harlow, et al . ; Pierce). Antibodies directed against N-(ABCDE) hepatitis polypeptides or fusion polypeptides (such as
  • N-(ABCDE) hepatitis particles can be further characterized by standard procedures including immunofluorescence microscopy, electron microscopy, Western blot analysis of proteins composing the particles, infection studies in animal and/or cell systems utilizing the partially purified particles, and sedimentation characteristics.
  • the N-(ABCDE) hepatitis particles can be disrupted to obtain N-(ABCDE) hepatitis genomes. Disruption of the particles can be achieved by, for example, treatment with detergents in the presence of chelating agents.
  • the genomic nucleic acid can then be further characterized. Characterization may include analysis of DNase and RNase sensitivity.
  • the strandedness and conformation (i.e., circular) of the genome can be determined by techniques known in the art, including visualization by electron microscopy and sedimentation characteristics.
  • RNA RNA
  • the isolated genomes also make it possible to sequence the entire genome whether it is segmented or not, and whether it is an RNA or DNA genome (using, for example RT-PCR, chromosome walking techniques, or PCR which utilizes primers from adjacent cloned sequences) . Determination of the entire sequence of a N-(ABCDE) hepatitis agent allows genomic organization studies and the comparison of the N-(ABCDE) hepatitis sequences to the coding and regulatory sequences of known viral agents.
  • N-(ABCDE) hepatitis agents provide the ability to screen for anti-hepatitis agents which inhibit the production of infectious N-(ABCDE) hepatitis agents: in particular, drugs that inhibit the replication of N- (ABCDE) hepatitis agents.
  • Cell culture and animal models allow the evaluation of the effect of such anti-hepatitis drugs on normal cellular functions and viability.
  • N-(ABCDE) hepatitis agent including, for example, small molecules, complex mixtures such as fungal extracts, and anti-sense oligonucleotides
  • Potential anti-viral agents are typically screened for anti-viral activity over a range of anti-viral agent concentrations.
  • the effect on N-(ABCDE) hepatitis agent replication and/or antigen production is then evaluated relative to the effect of the anti-viral agent on normal cellular function (DNA replication, RNA transcription, general protein translation, etc.).
  • the detection of the N-(ABCDE) hepatitis agent can be accomplished by the methods described in the present specification.
  • antibodies can be generated against the antigens of the present invention and these antibodies used in antibody-based assays (Harlow, et al .
  • N-(ABCDE) hepatitis antigens can be quantitated in culture using competition assays: polypeptides encoded by the cloned N-(ABCDE) hepatitis agent sequences can be used in such assays.
  • a recombinantly produced N-(ABCDE) hepatitis antigenic polypeptide is produced and used to generate a monoclonal or polyclonal antibody.
  • the recombinant N-(ABCDE) hepatitis polypeptide is labelled using a reporter molecule.
  • N-(ABCDE) hepatitis antigens The level of N-(ABCDE) hepatitis antigens in the sample is determined by comparison of levels of inhibition to a standard curve generated using unlabelled recombinant proteins at known concentrations.
  • the N-(ABCDE) hepatitis sequences of the present invention are particularly useful for the generation of polynucleotide probes/primers that may be used to quantitate the amount of N-(ABCDE) hepatitis nucleic acid sequences produced in a cell culture system.
  • probes labelled with reporter molecules can be used in standard dot-blot hybridizations or competition assays of labelled probes with infected cell nucleic acids.
  • polymerase chain reaction there are a number of methods using the polymerase chain reaction to quantitate target nucleic acid levels in a sample (Osikowicz, et al . ) .
  • Neutralizing antibodies can also be identified using the cell culture and animal model systems described above. For example, polyclonal or monoclonal antibodies are generated against the antigens of the present invention. These antibodies are then used to pre-treat sera before infection of cell cultures or animals. The ability of a single antibody or mixtures of antibodies to protect the cell culture or animal from infection is evaluated. For example, in cell culture and animals the absence of viral antigen and/or nucleic acid production serves as a screen. Further in animals, the absence of N-(ABCDE) hepatitis disease symptoms, e.g., elevated ALT values, is also indicative of the presence of neutralizing antibodies.
  • N-(ABCDE) hepatitis disease symptoms e.g., elevated ALT values
  • convalescent sera can be screened for the presence of neutralizing antibodies and then these sera used to identify N-(ABCDE) hepatitis agent antigens that bind with the antibodies.
  • the identified N-(ABCDE) hepatitis antigen is then recombinantly or synthetically produced.
  • the ability of the antigen to generate neutralizing antibodies is tested as above.
  • the antigen or antigens identified as capable of generating neutralizing antibodies can be used as a vaccine to inoculate test animals.
  • the animals are then challenged with infectious N-(ABCDE) hepatitis agents. Protection from infection indicates the ability of the animals to generate neutralizing antibodies that protect them from infection.
  • Vaccines can be prepared from one or more of the immunogenic polypeptides identified by the method of the present invention. Homologies between the isolated sequences from N-(ABCDE) hepatitis agents and other known viral proteins may provide information concerning the polypeptides that are likely to be candidates for effective vaccines. In addition, a number of computer programs can be used for to identify likely regions of isolated sequences that encode protein antigenic determinant regions (for example, Hopp, et al . ; "ANTIGEN,” Intelligenetics, Mountain View CA) .
  • Vaccines containing immunogenic polypeptides as active ingredients are typically prepared as injectables either as solutions or suspensions. Further, the immunogenic polypeptides may be prepared in a solid or lyophilized state that is suitable for resuspension, prior to injection, in an aqueous form. The immunogenic polypeptides may also be emulsified or encapsulated in liposomes. The polypeptides are frequently mixed with pharmaceutically acceptable excipients that are compatible with the polypeptides.
  • excipients include, but are not limited to, the following and combinations of the following: saline, water, sugars (such as dextrose and sorbitol) , glycerol, alcohols (such as ethanol [EtOH]), and others known in the art.
  • vaccine preparations may contain minor amounts of other auxiliary substances such as wetting agents, emulsifying agents (e . g. , detergents), and pH buffering agents.
  • emulsifying agents e g. , detergents
  • pH buffering agents such as sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate
  • adjuvants include, but are not limited to, the following: the group of related compounds including N-acetyl-muranyl- L-threonyl-D-isoglutamine and N-acetyl-nor-muranyl-L- alanyl-D-isoglutamine, and aluminum hydroxide.
  • the immunogenic polypeptides used in the vaccines of the present invention may be recombinant, synthetic or isolated from, for example, attenuated N-(ABCDE) hepatitis agent particles.
  • the polypeptides are commonly formulated into vaccines in neutral or salt forms.
  • Pharmaceutically acceptable organic and inorganic salts are well known in the art.
  • N-(ABCDE) hepatitis vaccines are parenterally administered, typically by subcutaneous or intramuscular injection.
  • Other possible formulations include oral and suppository formulations.
  • Oral formulations commonly employ excipients (e .g. , pharmaceutical grade sugars, saccharine, cellulose, and the like) and usually contain within 10-98% immunogenic polypeptide.
  • Oral compositions take the form of pills, capsules, tablets, solutions, suspensions, powders, etc., and may be formulated to allow sustained or long-term release.
  • Suppository formulations use traditional binders and carriers and typically contain between 0.1% and 10% of the immunogenic polypeptide.
  • multivalent vaccines against N-(ABCDE) hepatitis agents can be generated which are composed of one or more structural or non-structural viral-agent protein(s) .
  • These vaccines can contain recombinantly prepared N-(ABCDE) hepatitis agent polypeptides and/or polypeptides isolated from N-(ABCDE) hepatitis agent virions.
  • Vaccines may also be prepared from attenuated N- (ABCDE) hepatitis agent strains.
  • Such attenuated N- (ABCDE) hepatitis agents may be obtained utilizing the above described cell culture and/or animal model systems.
  • attenuated strains are isolated after multiple passages in vitro or in vivo . Detection of attenuated strains is accomplished by methods known in the art.
  • One method for detecting attenuated N-(ABCDE) hepatitis agents is the use of antibody probes against N-(ABCDE) hepatitis antigens, sequence-specific hybridization probes, or amplification with sequence-specific primers to screening in vivo or in vitro cultures.
  • Attenuated N-(ABCDE) hepatitis strains may be constructed based on the genomic information that can be obtained from the information presented in the present specification. Typically, a region of the infectious agent genome that encodes, for example, a polypeptide that is related to viral pathogenesis can be deleted. The deletion should not interfere with viral replication. Further, the recombinant attenuated N-(ABCDE) hepatitis agent construct allows the expression of an epitope or epitopes that are capable of giving rise to neutralizing antibodies against the N-(ABCDE) hepatitis agent.
  • the genome of the attenuated N-(ABCDE) hepatitis agent is then used to transform cells and the cells grown under conditions that allow viral replication. Such attenuated strains are useful not only as vaccines, but also as production sources of viral antigens and/or N-(ABCDE) hepatitis particles.
  • Hybrid particle immunogens that contain N-(ABCDE) hepatitis epitopes can also be generated.
  • the immunogenicity of N-(ABCDE) hepatitis epitopes may be enhanced by expressing the epitope in a eucaryotic systems (e . g. , mammalian or yeast systems) where the epitope is fused or assembled with known particle forming proteins.
  • hepatitis B surface antigen One such protein is the hepatitis B surface antigen.
  • Recombinant constructs where the N-(ABCDE) hepatitis epitope is directly linked to coding sequence for the particle forming protein will produce hybrid proteins that are immunogenic with respect to the N-(ABCDE) hepatitis epitope and the particle forming protein.
  • selected portions of the particle-forming protein coding sequence, which are not involved in particle formation may be replaced with coding sequences corresponding to N- (ABCDE) hepatitis epitopes.
  • regions of specific immunoreactivity to the particle-forming protein can be replaced by N-(ABCDE) hepatitis epitope sequences.
  • the hepatitis B surface antigen has been shown to be expressed and assembled into particles in the yeast Saccharomyces cerevisiea and in mammalian cells
  • the vaccines of the present invention are administered in dosages compatible with the method of formulation, and in such amounts that will be pharmacologically effective for prophylactic or therapeutic treatments.
  • the quantity of immunogen administered depends on the subject being treated, the capacity of the treatment subject's immune system for antibody synthesis, and the desired level of protection.
  • the amounts to be administered are usually determined by the administering health care professional.
  • N-(ABCDE) hepatitis vaccines of the present invention can be administered in single or multiple doses. Dosage regimens are also determined relative to the treatment subject's needs and tolerances. In addition to the N-(ABCDE) hepatitis immunogenic polypeptides, vaccine formulations may be administered in conjunction with other immunoregulatory agents, such as immunoglobins.
  • synthetic peptides can be generated which correspond to these polypeptides.
  • Synthetic peptides can be commercially synthesized or prepared using standard methods and apparatus in the art (Applied Biosystems, Foster City CA) .
  • oligonucleotide sequences encoding peptides can be either synthesized directly by standard methods of oligonucleotide synthesis, or, in the case of large coding sequences, synthesized by a series of cloning steps involving a tandem array of multiple oligonucleotide fragments corresponding to the coding sequence (Crea; Yoshio et al . ; Eaton et al.) . Oligonucleotide coding sequences can be expressed by standard recombinant procedures (Maniatis et al . ; Ausubel et al . ) .
  • One utility for the antigens obtained by the methods of the present invention is their use as diagnostic agents for hepatitis antibodies present in N-(ABCDE) sera, thereby indicating current or past infections in the individual; in particular, D19, Clone 17A, Clone IA.
  • the antigens of the present invention can be used singly, or in combination with each other, in order to detect single or multiple N-(ABCDE) hepatitis agents.
  • test serum is reacted with a solid phase reagent having a surface-bound antigen obtained by the methods of the present invention, e .g. , the D19 antigen.
  • the reagent After binding anti-N-(ABCDE) hepatitis antibody to the reagent and removing unbound serum components by washing, the reagent is reacted with reporter-labelled anti-human antibody to bind reporter to the reagent in proportion to the amount of bound anti-N- (ABCDE) hepatitis antibody on the solid support. The reagent is again washed to remove unbound labelled antibody, and the amount of reporter associated with the reagent is determined.
  • the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric or colorimetric substrate (Sigma, St. Louis, MO) .
  • the solid surface reagent in the above assay is prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, 96-well plate or filter material. These attachment methods generally include non-specific adsorption of the protein to the support or covalent attachment of the protein, typically through a free amine group, to a chemically reactive group on the solid support, such as an activated carboxyl, hydroxyl, or aldehyde group.
  • a second diagnostic configuration known as a homogeneous assay, antibody binding to a solid support produces some change in the reaction medium which can be directly detected in the medium.
  • homogeneous assays proposed heretofore include (a) spin- labelled reporters, where antibody binding to the antigen is detected by a change in reported mobility (broadening of the spin splitting peaks) , (b) fluorescent reporters. where binding is detected by a change in fluorescence efficiency, (c) enzyme reporters, where antibody binding effects enzyme/substrate interactions, and (d) liposome- bound reporters, where binding leads to liposome lysis and release of encapsulated reporter.
  • spin- labelled reporters where antibody binding to the antigen is detected by a change in reported mobility (broadening of the spin splitting peaks)
  • fluorescent reporters where binding is detected by a change in fluorescence efficiency
  • enzyme reporters where antibody binding effects enzyme/substrate interactions
  • liposome- bound reporters where binding leads to liposome lysis and release of encapsulated reporter.
  • the assay method involves reacting the serum from a test individual with the protein antigen and examining the antigen for the presence of bound antibody.
  • the examining may involve attaching a labelled anti-human antibody to the antibody being examined (for example from acute, chronic or convalescent phase) and measuring the amount of reporter bound to the solid support, as in the first method, or may involve observing the effect of antibody binding on a homogeneous assay reagent, as in the second method.
  • kits for carrying out the assay method just described.
  • the kit generally includes a support with surface-bound recombinant N-(ABCDE) hepatitis antigen (e.g., the D19 antigen, as above) , and a reporter-labelled anti-human antibody for detecting surface-bound anti-N-(ABCDE) antigen antibody.
  • N-(ABCDE) hepatitis antigen e.g., the D19 antigen, as above
  • reporter-labelled anti-human antibody for detecting surface-bound anti-N-(ABCDE) antigen antibody.
  • a third diagnostic configuration involves use of the anti-N-(ABCDE) hepatitis antibodies capable of detecting N-(ABCDE) hepatitis specific antigens.
  • the N-(ABCDE) hepatitis antigens may be detected, for example, using an antigen capture assay where N-(ABCDE) hepatitis antigens present in candidate serum samples are reacted with a N- (ABCDE) hepatitis specific monoclonal or polyclonal antibody. The antibody is bound to a solid substrate and the antigen is then detected by a second, different labelled anti-N-(ABCDE) hepatitis antibody.
  • Antibodies can be prepared, utilizing the peptides of the present invention, by standard methods. Antibodies that are substantially ' free of serum proteins which may affect reactivity can be generated (e . g. , affinity purification (Harlow et al . ) ) .
  • nucleic acid sequences obtained by the methods of the present invention are their use as diagnostic agents for hepatitis agent sequences present in N-(ABCDE) sera, thereby indicating infection in the individual.
  • Primers and/or probes derived from the coding sequences of the present invention in particular, D19, Clone 17A, Clone IA, and Clone 470-20-1 can be used singly, or in combination with each other, in order to detect single or multiple N-(ABCDE) hepatitis agents.
  • test serum is reacted under PCR or RT-PCR conditions using primers derived from, for example, 470-20-1 sequences.
  • Example 14 describes the use of polymerase chain amplification reactions, employing primers derived from the clones of the present invention, to screen different source material. The results of these amplification reactions demonstrate the ability of primers derived from the clones of the present invention (for example, 470-20- 1) , to detect homologous sequences by amplification reactions employing a variety of different source templates.
  • the amplification reactions in Example 14 included use of nucleic acids obtained directly from sera samples as template material.
  • probes can be derived from the N- (ABCDE) sequences of the present invention. These probes can then be labelled and used as hybridization probes against nucleic acids obtained from test serum or tissue samples.
  • the probes can be labelled using a variety of reporter molecules and detected accordingly: for example, radioactive isotopic labelling and chemiluminescent detection reporter systems (Tropix, Bedford, Mass.).
  • kits for carrying out the amplification/hybridization assay methods just described Such kits generally include either specific primers for use in amplification reactions or hybridization probes.
  • N-(ABCDE) hepatitis antigens of the present invention can be used in vaccine preparation.
  • antibodies generated against the polypeptide antigens of the present invention can be used for passive immunotherapy.
  • the anti-N-(ABCDE) hepatitis antibodies of the invention can be used as a means of enhancing an anti- N-(ABCDE) hepatitis immune response since antibody-virus complexes are recognized by macrophages.
  • the antibodies can be administered in amounts similar to those used for other therapeutic administrations of antibody. For example, pooled gamma globulin is administered at 0.02-0.1 ml/lb body weight during the early incubation of other viral diseases such as rabies, measles and hepatitis B to interfere with viral entry into cells.
  • antibodies reactive with the N-(ABCDE) hepatitis antigens can be passively administered alone or in conjunction with another anti-viral agent to a host infected with a N- (ABCDE) hepatitis agent to enhance the immune response and/or the effectiveness of an antiviral drug.
  • E. coli DNA polymerase I (Klenow fragment) was obtained from Boehringer Mannheim Biochemicals (Indianapolis, IN) .
  • T4 DNA ligase and T4 DNA polymerase were obtained from New England Biolabs (Beverly, MA) ; Nitrocellulose filters were obtained from Schleicher and Schuell (Keene, NH) .
  • Synthetic oligonucleotide linkers and primers were prepared using commercially available automated oligo ⁇ nucleotide synthesizers. Alternatively, custom designed synthetic oligonucleotides may be purchased from commercial suppliers. cDNA synthesis kit and random priming labeling kits were obtained from Boehringer-Mannheim Biochemical (BMB, Indianapolis, IN) or GIBCO/BRL (Gaithersburg, MD) .
  • Standard molecular biology and cloning techniques were performed essentially as previously described in Ausubel, et al . , Sambrook, et al . , and Maniatis, et al. Common manipulations involved in polyclonal and monoclonal antibody work, including antibody purification from sera, were performed by standard procedures (Harlow, et al . ) . Pierce or Promega (Madison, WI) were sources of many antibody reagents.
  • Plates are aspirated and blocked with 300 uL 10% NGS and incubated at 37°C for 1 hr.
  • Antisera were diluted in 0.1 M PBS, pH 7.2. The desired dilution(s) of antisera (0.1 mL) were added to each well and the plate incubated 1 hours at 37°C. The plates was then washed 5 times with PBS 0.5% "TWEEN- 20".
  • HRP Horseradish peroxidase conjugated goat anti- human antiserum
  • the reagent consists of 50 mL 0.05 M citric acid, pH 4.2, 0.078 mL 30% hydrogen peroxide solution and 15 mg ABTS. 0.1 mL of the substrate was added to each well, then incubated for 30 min at room temperature. The reaction was stopped with the addition of 0.050 mL 5% SDS (w/v) . The relative absorbance is determined at 410 nm.
  • the sera samples in the Table 1 were tested in standard ELISA assays using a number of known hepatitis antigens including the C100 Abbott test kit antigen (Abbott Laboratories, N. Chicago, IL) and HCV and HEV individual antigens.
  • the 409-1-1 and the 33u antigens are from HCV NS3, antigen 36 from HCV NS5. All HCV epitopes, except antigen 36, were expressed as a fusion protein of glutathione-S-transferase (Smith, et al . ) .
  • the original PNF 2161 inoculum was positive with HCV antigens.
  • the My131 inoculated with PNF2161 tested negative for the presence of HCV by the RT-PCR assay, PCR using non-coding region HCV primers, and by ELISA with HCV antigens.
  • HCV nucleic acid was also tested using a reverse transcription polymerase chain reaction (Kawasaki, et al . ; Wang, et al . ) .
  • HCV epitopes have been disclosed in Houghten, et al . , (C-100) and Reyes, et al . , (WO 91/15516,
  • each undiluted serum (My 620 and My 670) was pelleted by centrifugation at 40,000 rpm in a type 70.1 rotor for 2 hours at 4°C.
  • Each resulting pellet (by either of the above methods) was extracted for RNA and DNA.
  • Half of the nucleic acids were converted to cDNA with random primer and reverse transcriptase after denaturation. The other half of each was converted to DNA with random primer and Klenow.
  • the pellet was treated with a solution containing 4M guanidine isothiocyanate, 0.18% 2- mercaptoethanol, and 0.5% sarcosyl.
  • the treated pellet was extracted several times with acidic phenol-chloroform alcohol, and the RNA was precipitated with ethanol. This solution was held at -70°C for approximately 10 minutes and then spun in a microfuge at 4°C for 10 minutes.
  • nucleic acid pellet was treated with RNase-free DNase I (Ausubel, et al . ) prior to first strand synthesis; My 131 and My 190 sera were not treated with DNase.
  • T4 DNA polymerase was added to the mixture to maximize the number of blunt-ends of cDNA molecules.
  • the reaction mixture was incubated at room temperature for 10 minutes.
  • the reaction mixture was extracted with phenol/chloroform and chloroform isoamyl alcohol.
  • the cDNA was precipitated by the addition of two volumes of 100% ethanol and chilling at -70°C for 15 minutes.
  • the cDNA was collected by centrifugation, the pellet washed with 70% ethanol and dried under vacuum.
  • the cDNA pellet was resuspended in 12 ⁇ l distilled water.
  • Linker AB a double strand linker comprised of SEQ ID NO:44 and SEQ ID NO:45, where SEQ ID NO:45 is in a 3' to 5' orientation relative to SEQ ID NO:44 — as a partially complementary sequence to SEQ ID NO:44
  • 1 ⁇ l T4 DNA ligase 1 ⁇ l T4 DNA ligase.
  • the cDNA and linker were mixed at a 1:100 molar ratio in the presence of 0.3 to 0.6 Weiss units of T4 DNA ligase.
  • the reaction was incubated at 14°C overnight. The following morning the reaction was incubated at 70°C for three minutes to inactivate the ligase.
  • Thermus aquaticus DNA polymerase (Taq polymerase).
  • the reaction mixture was heated to 94°C for 30 sec for denaturation, allowed to cool to 50°C for 30 sec for primer annealing, and then heated to 72°C for 0.5-3 minutes to allow for primer extension by Taq polymerase.
  • the amplification reaction involving successive heating, cooling, and polymerase reaction, was repeated an additional 25-40 times with the aid of a Perkin-Elmer Cetus DNA thermal cycler (Mullis; Mullis, et al . ; Reyes, et al . , 1991).
  • the solution was then phenol/chloroform, chloroform/isoamyl alcohol extracted and precipitated with two volumes of ethanol.
  • the .Eco.RJ-digested cDNA preparations were ligated into lambda gtll (Promega) .
  • the ligation reaction tubes were placed at 14°C overnight (12-18 hours) .
  • the ligated cDNA was packaged the following morning by standard procedures using a lambda DNA packaging system (GIGAPAK, Stratagene, LaJolla, CA) , and then plated at various dilutions to determine the titer.
  • GOGAPAK lambda DNA packaging system
  • a standard X-gal blue/white assay was used to determine recombinant frequency of the libraries (Miller; Maniatis et al . ) .
  • Percent recombination in each library was also determined as follows. A number of random clones were selected and corresponding phage DNA isolated. Polymerase chain reaction (Mullis; Mullis, et al . ) was then performed using isolated phage DNA as template and lambda DNA sequences, derived from lambda sequences flanking the EcoRI insert site for the cDNA molecules, as primers. The presence or absence of insert was evident from gel analysis of the polymerase chain reaction products.
  • cDNA-insert phage libraries were generated from sera samples My 131, My 190, My 620, My 670, DEN, SCH, JFA and PNF 2161.
  • the cDNA-insert phage libraries generated from sera samples My 131, My 190, DEN, SCH, JFA and PNF 2161 have been deposited with the American Type Culture Collection, 12301 Parklawn Dr., Rockville MD 20852, and have been assigned the following deposit designations: MY 131 cDNA source, ATCC 75273; PNF 2161 cDNA source, ATCC 75268; JFA cDNA source 75272; SCH cDNA source, ATCC 75283; DEN cDNA source, ATCC 75417; and MY 190, cDNA source, to be assigned.
  • the cDNA- insert phage libraries generated from sera samples My 620 and My 670 can be obtained from Genelabs Technologies, Inc., 505 Penobscot Dr., Redwood City, CA 94063.
  • A. My 131 and My 190 Libraries (i) Isolation of Nucleic Acids. Each sera, My 131 and My 190, was pelleted as described above. The resulting pellet was resuspended in 0.1 M NaCl, 50 mM Tris, pH 8, 1 mM EDTA, 0.5% SDS and treated with Proteinase K at a final concentration of 1 mg/ml. Nucleic acids were precipitated, after phenol/chloroform and chloroform/isoamyl alcohol extractions, by the addition of two volumes of ice-cold ethanol. This solution was held at -70°C for approximately 10 minutes and then spun in a microfuge at 4°C for 10 minutes.
  • the resulting pellet was resuspended in 100 ⁇ l of sterile TE (Maniatis, et al . ) .
  • sterile TE Maniatis, et al .
  • the solution was held at -70°C for at least 10 minutes.
  • the nucleic acid pellet was recovered by centrifugation in a microfuge at 12,000 x g for 15 minutes at 5°C.
  • the pellet was washed in 70% ethanol, dried under vacuum, and resuspended in a minimum volume of TE.
  • the reaction mixture was heated to 95°C for 5 minutes to denature the DNA, and 1 ⁇ l of Klenow (1.5 u) was added to start the reaction upon cooling of the mixture.
  • the reaction was placed at 37°C and typically carried out for 30 minutes.
  • the reacted was stopped by the heat-inactivation of the Klenow enzyme at 65°C for 10 minutes. Blunt ends for the DNA molecules were generated by the treatment with the DNA polymerase as described previously.
  • the random primed DNA mixture was ligated to linkers, PCR amplified and cloned in lambda gtll vectors as described in Example 2.
  • DNA libraries from sera JFA, PNF 2161, SCH, DEN, My 620 and My 670 were prepared essentially as described for the My 131 and My 190 libraries, except that, prior to random primer DNA synthesis, the nucleic acid samples were treated with DNase-free RNase (Boehringer Mannheim) (Ausubel, et al . ; Maniatis, et al . ) for JFA and PNF2161 libraries.
  • DNase-free RNase Boehringer Mannheim
  • the isolated nucleic acid was used as template for random primed DNA synthesis reactions, as described above.
  • the samples were precipitated and resuspended as described above.
  • Normal human serum DNA was extracted from a healthy donor from Stanford blood bank, subjected to random priming and then ligated to C/D primer linker (nucleotide sequences of oligonucleotide C: SEQ ID NO:8 and of oligonucleotide D: SEQ ID NO:19).
  • the resulting normal human DNA was 5' end-labelled with biotin using a biotinylated primer (at 1 ⁇ M) in a polymerase chain reaction.
  • the reaction was performed as described in Example 2C except that the mixture was heated to 94°C for one minute, allowed to cool to 50°C for two minutes, and then heated to 72°C for 3 minutes and that the cycle was repeated 30 times.
  • SISPA- amplified SCH DNA was incubated with 5 ⁇ g of the biotinylated normal human DNA in 50 ⁇ l of 0.12 M sodium phosphate buffer (pH 7.0) at 95°C for 5 minutes. The tube was then placed in a water bath heated to 55°C for 4 days. Streptavidin-conjugated paramagnetic particles ("DYNABEADS"; Dynal Inc., Lake Success, NY) suspended in 50 ⁇ l of 2x binding solution (2 M NaCl, 1 mM EDTA, 10 mM Tris-HCl, pH 7.5) were added to the solution containing the DNA, and the mixture was incubated at room temperature for 15 minutes.
  • 2x binding solution 2 M NaCl, 1 mM EDTA, 10 mM Tris-HCl, pH 7.5
  • DNA-insert phage libraries were generated from sera samples My 131, My 190, My 620, My 670, DEN, SCH, JFA and PNF 2161.
  • the DNA-insert phage libraries generated from sera samples My 131, My 190, DEN, SCH, JFA and PNF 2161 have been deposited with the American Type Culture Collection, 12301 Parklawn Dr., Rockville MD 20852, and have been assigned the following deposit designations: MY 131 DNA source, ATCC 75270; PNF 2161 DNA source, ATCC 75269; JFA DNA source, ATCC 75271; SCH DNA source, ATCC 75282; DEN DNA Source, ATCC 75418, and MY 190, DNA source, ATCC 75284.
  • the DNA-insert phage libraries generated from sera samples DEN, My 620 and My 670, as well as subtracted SCH DNA libraries can be obtained from Genelabs Technologies, Inc. 505 Penobscot Dr., Redwood City, CA 94063.
  • the lambda gtll libraries generated in Examples 1 and 2 were immunoscreened for the production of antigens recognizable by the five sera, or corresponding sera, from which the libraries were generated.
  • the phage were plated for plaque formation using the Escherichia coli bacterial plating strain E. coli KM392 (Kevin Moore, DNAX, Palo Alto, CA) .
  • E. coli Y1090R- may be used.
  • the fusion proteins expressed by the lambda gtll clones were screened with serum antibodies essentially as described by Ausubel, et al . Each library was plated at approximately 2 x 10 4 phages per 150 mm plate. Plates were overlaid with nitrocellulose filters overnight. Filters were washed with TBS (10 mM, Tris pH 7.5; 150 mM NaCl) , blocked with AIB (TBS buffer, 10 mM Tris, pH 8.0, 150 mM NaCl; with 1% gelatin) and incubated with a primary antibody diluted 100 times in AIB.
  • TBS 10 mM, Tris pH 7.5; 150 mM NaCl
  • AIB TBS buffer, 10 mM Tris, pH 8.0, 150 mM NaCl; with 1% gelatin
  • DNA sources normal human peripheral blood lymphocyte
  • Escherichia coli KM392 genomic DNA (Ausubel, et al . ; Maniatis, et al . ; Sambrook, et al . ) .
  • Ten micrograms each of human lymphocyte DNA and normal mystax DNA, and 2 micrograms of E. coli DNA were digested with EcoRI and Hindlll.
  • the restriction digestion products were electrophoretically fractionated on an agarose gel (Ausubel, et al . ) and transferred to nylon or nitrocellulose membranes (Schleicher and Schuell, Keene, NH) as per the manufacturer's instructions.
  • Probes from the immunoreactive clones were prepared as follows. Each clone was amplified using primers corresponding to lambda gtll sequences that flank the EcoRI cloning site of the gtll vector. Amplification was carried out by polymerase chain reactions utilizing each immunoreactive clones as template. The resulting amplification products were digested with EcoRI, the amplified fragments gel purified and eluted from the gel (Ausubel, et al . ) . The resulting amplified fragments, derived from the immunoreactive clones, were then random prime labelled (Boehringer Mannheim) using 32 P-dNTPs.
  • the random primed probes were then hybridized to the above-prepared nylon membrane to test for hybridization of the insert sequences to the control DNAs. Typically, inserts that hybridized with any of the control DNAs were removed from consideration.
  • a probe derivative from a human C-kappa gene fragment (Hieter) was used as single gene copy control for human DNA and a E . coli polymerase gene fragment was similarly used for E. coli DNA.
  • the putative viral particles were then pelleted by centrifugation at 40,000 rpm in the Ti70.l rotor (approximately 110,000 x g) at 4°C for 2 hours, and RNA was extracted using the acid guanidinium phenol technique ("TRI REAGENT", Molecular Research Center, Cincinnati, OH) , and alcohol-precipitated using glycogen as a carrier to improve recovery.
  • the purified nucleic acid was dissolved in an RNase-free buffer containing 2 mM DTT and 1 U/ml recombinant RNasin.
  • RNA was passed through a "CHROMA SPIN" 100 gel filtration column (Clontech, Palo Alto, CA) to remove small molecular weight impurities.
  • cDNA was synthesized using a Boehringer-Mannheim cDNA synthesis kit (Boehringer-Mannheim, Indianapolis, IN) . After cDNA synthesis the PNF cDNA was ligated to a 50 to 100 fold excess of KL-l/KL-2 SISPA linkers (SEQ ID NO:64, SEQ ID NO:65, respectively) and amplified for 35 cycles using the primer KL-1.
  • the 470 extension clones were generated by anchored PCR of a 1 ⁇ l aliquot from a 10 ⁇ l ligation reaction containing EcoRI digested (dephosphorylated) lambda gtll arms (1 ⁇ g) and EcoRI digested PNF cDNA (0.2 ⁇ g) .
  • PCR amplification (40 cycles) of the ligation reaction was carried out using the lambda gtll reverse primer in combination with either 470-20-77F (SEQ ID NO:25) or 470-20-1-211R (SEQ ID NO:26). All primer concentrations for PCR were 0.2 ⁇ M.
  • the amplification products (9 ⁇ l/100 ⁇ l) were separated on a 1.5% agarose gel, blotted to "NYTRAN" (Schleicher and Schuell, Keene, NH) , and probed with a digoxygenin labelled oligonucleotide probe specific for 470-20-1.
  • the digoxygenin labeling was performed according to the manufacturer's recommendations using terminal transferase (Boehringer-Mannheim) . Bands that hybridized were gel-purified, cloned into the "TA
  • Percent recombinant clones in the indicated ⁇ gtll library as determined by blue/white plaque assay and confirmed by PCR amplification of randomly selected clones.
  • Antisera source used for the immunoscreening of each indicated library.
  • Percent recombinant clones in the indicated ⁇ gtll library as determined by blue/white plaque assay and confirmed by PCR amplification of randomly selected clones.
  • Antisera source used for the immunoscreening of each indicated library.
  • Seven candidate clones were found to have open reading frames in phase with the ⁇ -galactosidase - 427- 7-1, 428-2-3, 428-7-3, 428-3-1, 430-4-8, 430-2-1 and 430-3-4.
  • Seven primary candidate clones were found having open reading frames continuous with the 3-galactosidase - D5, D6, D12, D13, D19, D44, and D76. Eleven additional clones contained open reading frames greater than 15 amino acids in length. The are: D20, D24, R27, D30, D31-2, D48, and D64.
  • MY 73 and MY 136 were initially infected with 1 ml of 10% liver homogenate of MY 131 treated with CHC1 3 . 100 days after infection, MY 73 and MY 136 were cross challenged with GB agent; MY 242, infected with 10% liver homogenate of MY 131 (not treated with CHC1 3 ) , was cross challenged with GB agent after 100 days; MY 190 and MY 88 are, respectively, the acute phase and convalescent phase sera for GB agent; MY 162 is a normal mystax serum; MY 131 is the acute phase mystax serum for PNF2161; and PNF2161 is a chronic human hepatitis serum. "Pre-GB” means 100 days after inoculation with MY 131 liver homogenate, that is, convalescent phase sera that corresponds to the MY 131 inoculum. EXAMPLE 9
  • the D19 clone encoded antigen was immunoscreened against nine additional mystax serum samples.
  • the clone was immunoreactive with 2/9 of the sera.
  • the D6 and D19 sequences were subcloned into the bacterial expression vector, pGEX-GLI.
  • the pGEX-GLI is a modification of the pGEX-1 vector of Smith, et al . , which involved the insertion of a thrombin cleavage sequence in-frame with the glutathione-S-transferase protein (GST: sj26 coding sequence) and addition of
  • the WcoJ primers in the amplified fragment allow in-frame fusion of D6 or D19 coding sequence to the sj26-thrombin coding sequences.
  • the D19 coding sequence insert was generated by the polymerase chain reaction using PCR primers specific for each insert. Typically, the 5' primer contains a Ncol restriction site and the 3' primer contains stop-codons followed by a BamHI restriction site.
  • the D19-5' primer was D19-NF (SEQ ID NO:68).
  • the D19-3' primer was D19-RF (SEQ ID NO:69).
  • the generated PCR product was digested with Ncol and BamHI and gel purified.
  • D6 coding sequence insert was prepared in a similar fashion.
  • the D6-5' primer was D6-NF (SEQ ID NO:66) and the D6-3' primer was D6-BR (SEQ ID NO:67).
  • the pGEX-GLI vector was digested with Ncol and BamHI and the linear vector isolated.
  • Manipulations were carried out for both coding sequence inserts essentially as described below for the D19 insert sequence.
  • the Ncol /BamHI D19 fragment was ligated to the linear pGEX-GLI vector.
  • the ligation mixture was transformed into E . coli and ampicillin resistant colonies were selected. Plasmids were isolated from the ampicillin resistant colonies and analyzed by restriction enzyme digestion. One of the candidate clones was designated pGEX-GLI-D19.
  • E. coli strain JM101 was transformed with pGEX- GLI-D19 and was grown at 37°C overnight. DNA was prepared from randomly-picked colonies. The presence of the insert coding sequence was confirmed by (i) restriction digest mapping and (ii) hybridization screening using labelled D19 inserts (i.e.. Southern analysis) .
  • a D19 clone was identified, see above, and grown overnight.
  • the overnight culture was diluted 1:10 with LB medium containing ampicillin and grown for one hour at 37°C.
  • the overnight culture was diluted 1:100 and grown to CD of 0.5-1.0 before addition of IPTG (isopropylthio-j8-galactoside) .
  • IPTG isopropylthio-j8-galactoside
  • Bacterial cells were harvested by centrifugation and resuspended in 1/100 culture volume of MTPBS (150 mM NaCl, 16 mM Na 2 HP0 4 , 4 mM NaH 2 P0 4 ) . Cells were lysed by lysozyme, sonication or French press, and lysates cleared of cellular debris by centrifugation.
  • MTPBS 150 mM NaCl, 16 mM Na 2 HP0 4 , 4 mM NaH 2 P0 4
  • IPTG-induced cultures of pGEX-GLI-vector alone were analyzed by SDS-polyacrylamide gel electrophoresis followed by Western blotting, as described below.
  • the extracts can be concentrated by ultrafiltration using, for example, a "CENTRICON 10" filter.
  • the fusion proteins were partially purified over a glutathione agarose affinity column as described in detail by Smith, et al .
  • 100 ml cultures were grown overnight. The cultures are diluted to 1 liter, and the cells grown another hour at 37°C. Expression of the fusion proteins was induced using IPTG. The induced cultures were grown at 37°C for 3.5 hours. Cells were harvested and a sonicator was used to lyse the cells. Cellular debris was pelleted and the clear lysate was loaded onto a glutathione "SEPHAROSE" column. The column was washed with several column volumes. The fusion protein was eluted from the affinity column with reduced glutathione and dialyzed.
  • the membranes were incubated with the test serum (for example, Mystax 88 or Ruf serum) and washed.
  • the membranes were then incubated with a labelled antibody suitable for detection of binding of antibodies from the test sera: for example, alkaline phosphatase- conjugated goat anti-human antibody (Mystax 88) .
  • a labelled antibody suitable for detection of binding of antibodies from the test sera for example, alkaline phosphatase- conjugated goat anti-human antibody (Mystax 88) .
  • Excess goat anti-human IgG antibody was removed from the membranes (Ausubel et al . ; Harlow et al . ) and the membranes colori etrically developed.
  • Figure 3 illustrates the results of a Western blot analysis of the membrane containing crude lysates of the following samples: induced ( Figure 3, lane 1) and un-induced ( Figure 3, lane 2) pGEX-GLI-D6; induced ( Figure 3, lane 3) and un-induced ( Figure 3, lane 4) pGEX-GLI-D19.
  • the test serum was a 1:100 dilution of N-(ABCDE) mystax (My 88) serum.
  • Figures 4A and 4B illustrates Western blots of the partially purified proteins using mystax (My 88) serum ( Figure 4A) and N-(ABCDE) human (Ruf) serum ( Figure 4B) .
  • the partially purified proteins include D6 and D19 fusion proteins.
  • the sj26 antigen was used as a control.
  • the membranes treated with infected sera showed specific immunoreactivity with the N-(ABCDE) fusion proteins and Ruf serum reacted weakly to Sj26.
  • the pGEX-GLI-D19 is considered negative with the human serum.
  • Percent recombinant clones in the indicated ⁇ gtll library as determined by blue/white plaque assay and confirmed by PCR amplification of randomly selected clones.
  • Clones 17A and IA have both been shown to have exogenous, non-human insert sequences. Both clones contain a single coding sequence continuous with the open reading frame of -galactosidase in the lambda gtll vector.
  • Figure 5B shows a Western blot analysis of the Figure 5A polyacrylamide gel transferred to a membrane and probed with JFA serum.
  • both the SJ26-17A and SJ26-1A fusion proteins are immunoreactive with JFA serum; however, equivalent amounts of Sj26 protein are not immunoreactive.
  • the arrows indicate the locations of the SJ26-17A and SJ26-1A fusion proteins, first and second panels, respectively.
  • Figure 5C shows the Western blot analysis of Figure 5A polyacrylamide gel transferred to a membrane and probed with normal human serum. As can be seen in Figure 5C, IA and 17A proteins are not immunoreactive to normal serum.
  • D Isolation of Overlapping Clones to Clones 17A and IA
  • a JFA DNA library was generated in "LAMBDA ZAPII" (Stratagene, LaJolla, CA) using JFA random-primed SISPA DNA, prepared essentially as described in Example 3, except that the amplified DNA was digested with WotJ instead of EcoRI.
  • Undigested "LAMBDA ZAPII” DNA was cut with NotI and then treated with calf intestinal phosphatase (CIP) to prevent re-ligation of phage DNA ends.
  • CIP calf intestinal phosphatase
  • a ligation reaction to insert JFA SISPA DNA fragments into phage DNA at the NotJ site was performed essentially as described in Example 2D.
  • the ligated cDNA was packaged the following morning by standard procedures using a lambda DNA packaging system
  • the packaged phage were used to infect E . coli strain PLK-A' .
  • a titer for the recombinant DNA phage library was determined by standard methods.
  • the hybridization-positive clones were screened with oligonucleotides located at 5'- or 3'-ends of clone 17A.
  • oligonucleotides located at 5'- or 3'-ends of clone 17A were screened with oligonucleotides located at 5'- or 3'-ends of clone 17A.
  • the oligonucleotides were end-labelled with T4 polynucleotide kinase and 7 ⁇ 32 P-ATP (Maniatis, et al . )
  • the screening procedure using oligonucleotides complementary to clone 17A resulted in l positive and 7 negatives.
  • the clone that showed a positive hybridization to the 3'-end primer contained an insert of about 400 base pairs as estimated by gel electrophoresis.
  • This clone was designated clone WT54.
  • the insert of WT54 was sequenced and contained a 210 base pair overlap with 17A which extends 119 base pairs from the 3'-end of 17A.
  • the sequence of this insert is presented as SEQ ID NO:58.
  • Figure 6 schematically illustrates the overlap between clone 17A and WT54.
  • the 17A clones that gave a negative hybridization result with the oligonucleotide primers were rescreened.
  • the probe used for rescreening was a PCR- generated clone 17A insert generated from 5'-and 3'-end oligonucleotide primers (SEQ ID NO:70 and SEQ ID NO:71) , and digested with Ncol and BamHI prior to labelling. This procedure resulted in 6 positives and 1 negative. All 6 positive clone inserts were sequenced and corresponded to internal 17A sequences.
  • WT54 Clone The WT54 clone was characterized for its ability to hybridize to the following control DNA sources: normal human peripheral blood lymphocyte DNA (purchased from Stanford Blood Bank) , normal mystax liver DNA (Centers for Disease Control) , and Escherichia coli KM392 genomic DNA (Ausubel, et al . ; Maniatis, et al.; Sambrook, et al.) . Ten micrograms each of human lymphocyte DNA and normal mystax DNA, and 2 micrograms of E. coli DNA were digested with EcoRI and Hindlll. The restriction digestion products were electrophoretically fractionated on an agarose gel
  • Radiolabelled clone WT54 was prepared as follows. Oligonucleotide 5'- and 3'- primers were synthesized in the region of clone WT54 that does not overlap with clone 17A.
  • the 5'-primer is WT54-590F (SEQ ID NO:59) and the 3'-primer is WT54-684R (SEQ ID NO:60).
  • the primers were used to amplify the region between base pairs 590 and 684 by PCR.
  • the resulting amplified fragment was then labelled by the random-priming method (Boehringer Mannheim) using 32 P-dNTPs and then used as a probe.
  • the labelled probe was then hybridized to the above-prepared nylon membrane to test for hybridization of the insert sequences to the control DNAs.
  • the probe did not hybridize to human, E. coli or mystax genomic DNA.
  • the following primers were selected, prepared and used in amplification reactions: a 5'-primer unique to clone 17A sequences, i.e., 17A sequences that do not overlap WT54 sequences; and a 3'- primer unique to clone WT54 sequences, i . e . , WT54 sequences that do not overlap 17A sequences.
  • the 5'- primer is 17A-215F ( Figure 7, A — SEQ ID NO:61) and the 3'-primer is WT54-684R ( Figure 7, B — SEQ ID NO:60).
  • the primers were used to amplify the putative linked region between base pairs 215 and 684 by PCR using JFA SISPA DNA and serum DNA as template.
  • the resulting amplified product was then amplified using internal nested primers, 17A-258F (Figure 7, C — SEQ ID NO:62) and WT54 647R (Figure 7, D — SEQ ID NO:63).
  • the final PCR product was probed with a labelled-oligonucleotide specific for a non-overlapping 17A insert sequence, 17A-312F ( Figure 7, Probe 1 — SEQ ID NO:52), and a labelled-oligonucleotide specific to a non-overlapping WT54 sequence, WT54-592F ( Figure 7, Probe 2 — SEQ ID NO:53).
  • the unsubtracted and subtracted SCH DNA libraries generated from SCH serum in lambda gtll, were screened with convalescent SCH serum as described in Example 4.
  • the DNA clones obtained from the unsubtracted library were designated "SU”.
  • the DNA clones from the subtracted DNA library of SCH were designated "SC”. The results of the screening are presented in Table 10.
  • EXAMPLE 12 Isolation of D19 Fusion Protein Sepharose 4B beads conjugated with anti-?- galactosidase is purchased from Promega. The beads are packed in 2 ml column and washed successively with phosphate-buffered saline with 0.02% sodium azide and 10 ml TX buffer (10 mM Tris buffer, pH 7.4, 1% aprotinin) .
  • Lysogens infected with gtll/D19 are used to inoculate 500 ml of NZYDT broth.
  • the culture is incubated at 32°C with aeration to an O.D. of about .2 to .4, then brought to 43°C quickly in a 43°C water bath for 15 minutes to induce gtll peptide synthesis, and incubated further at 37°C for 1 hour.
  • the cells are pelleted by centrifugation, suspended in 10 ml of lysis buffer (10 mM Tris, pH 7.4 containing 2% "TRITON X-100" and 1% aprotinin added just before use.
  • the resuspended cells are frozen in liquid nitrogen, then thawed, resulting in substantially complete cell lysis.
  • the lysate is treated with DNase I to digest bacterial and phage DNA, as evidenced by a gradual loss of viscosity in the lysate.
  • Non-solubi- lized material is removed by centrifugation.
  • the clarified lysate material is loaded on the Sepharose column, the ends of the column closed, and the column placed on a rotary shaker for 2 hrs. at room temperature and 16 hours at 4°C. After the column settles, it is washed with 10 ml of TX buffer.
  • the fused protein is eluted with 0.1 M carbonate/bicarbonate buffer, pH 10. Typically, 14 ml of the elution buffer is passed through the column, and the fusion protein is eluted in the first 4-6 ml of eluate.
  • the eluate containing the fusion protein is concentrated in "CENTRICON-30" cartridges (Amicon, Danvers, Mass.).
  • the final protein concentrate is resuspended in, for example, 400 ⁇ l PBS buffer. Protein purity is analyzed by SDS-PAGE.
  • EXAMPLE 13 Preparation of Anti-D19 Antibody Expression of a glutathione S-transferase fused protein (Sj26 fused protein) containing the D19 peptide antigen was achieved in E. coli strain JM101 (above) .
  • the fusion protein is isolated from lysed bacteria, and isolated by affinity chromatography on a column packed with glutathione-conjugated beads, according to published methods (Smith, et al . ) .
  • the purified SJ26/D19 fused protein is injected subcutaneously in Freund's adjuvant in a rabbit. Approximately 1 mg of fused protein is injected at days 0 and 21, and rabbit serum is typically collected at 6 and 8 weeks. A second rabbit is similarly immunized with purified Sj26 protein obtained from control bacterial lysate.
  • Minilysates from the following bacterial cultures are prepared: (1) KM392 cells infected with pGEX and pGEX containing the D19 insert; and (2) cells infected with lambda gtll containing the D19 insert.
  • the minilysates and a commercial source /3-galactosidase are fractionated by SDS-PAGE, and the bands transferred to nitrocellulose filters for Western blotting. Summarizing the expected results, serum from control (Sj26) rabbits is immunoreactive with each of the Sj26 and Sj26 fused protein antigens.
  • Serum from the animal immunized with SJ26/D19 fused protein is reactive with all Sj-26 and ?-gal fusion proteins containing D19 coding sequences, indicating the pre ⁇ sence of specific immunoreaction with the D19 antigen. None of the sera are expected to be immunoreactive with ?-galactosidase.
  • Anti-D19 antibody present in the sera from the animal immunized with the SJ26/D19 is purified by affinity chromatography (using the D19 ligand, essentially as described above in Example 12 for the anti-yff-galactosidase antibody.
  • PCR DETECTION OF N(ABCDE) HEPATITIS SEQUENCES A polymerase chain reaction testing algorithm was devised first to verify exogenicity with respect to several genomic DNAs which could have been inadvertently cloned during library construction, then to test for the presence of the cloned sequence in the cloning source and related specimen materials.
  • specimens including SISPA-amplified nucleic acids and nucleic acids extracted from the primary source, and nucleic acids extracted from related source materials (e . g. , from animal passage studies) , were tested.
  • Amplification amplified cDNA and DNA were used as templates (Example 2) .
  • Sequence-specific primers designed from selected cloned sequences were used to amplify DNA fragments of interest from the templates.
  • the templates were the SISPA-amplified samples used in the cloning manipulations.
  • amplification primers 470-20-1-77F (SEQ ID NO:25) and 470-20-1-211R (SEQ ID NO:26) were selected from the clone 470-20-1 sequence (SEQ ID NO:106).
  • Positive control DNA used in the amplification reactions was previously amplified PCR product whose concentration was estimated by the Hoechst 33258 fluorescence assay, or, alternatively, purified plasmid DNA containing the cloned inserts of interest.
  • the 470-20-1 specific signal was detected in cDNA amplified by PCR from SISPA-amplified PNF2161. Negative control reactions were nonreactive, and positive control DNA templates were detected.
  • genomic PCR refers to testing for the presence of specific sequences in genomic DNA from relevant organisms.
  • genomic DNAs as follows:
  • E. coli (10-100 ng/rxn.)
  • yeast (10-100 ng/rxn.)
  • E. coli genomic DNA as a frequent contaminant of commercial enzyme preparations, is tested.
  • Yeast is also tested, as a ubiquitous organism, whose DNA can contaminate reagents and thus, be cloned.
  • a negative control i.e., buffer or water only
  • positive controls to include approximately 10 5 c/rxn.
  • Amplification conditions vary, as may be determined for individual sequences, but follow closely the following standard PCR protocol: PCR was performed in reactions containing 10 mM Tris, pH 8.3, 50 mM KCl, 1.75 mM MgCl 2 , 1.0 uM each primer, 200 uM each dATP, dCTP, and dGTP, and 300 urn dUTP, 2.5 units Taq DNA polymerase, and 0.2 units uracil-N-glycosylase per 100 ul reaction.
  • PCR reagents were assembled, and amplification reactions were constituted, in a specially-designated laboratory maintained free of amplified DNA. As a further barrier to contamination by amplified sequences and thus compromise of the test by "false positives," the PCR was performed with dUTP replacing TTP, in order to render the amplified sequences biochemically distinguishable from native DNA.
  • uracil-N-glycosylase was included in all genomic PCR reactions. Upon conclusion of thermal cycling, the reactions were held at 72°C to prevent renaturation of uracil-N-glycosylase and possible degradation of amplified U-containing sequences.
  • a Hot Start PCR was performed, using standard techniques ("AMPLIWAX", Perkin-Elmer Biotechnology, Norwalk, CT; alternatively, manual techniques were used) , in order to make the above general protocol more robust for amplification of diverse sequences, which ideally require different amplification conditions for maximal sensitivity and specificity.
  • Detection of amplified DNA was performed by hybridization to specific oligonucleotide probes located internal to the two PCR primer sequences and having no or minimal overlap with the primers. In some cases, direct visualization of electrophoresed PCR products was performed, using ethidium bromide fluorescence, but probe hybridization was in each case also performed, to help ensure discrimination between specific and non-specific amplification products. Hybridization to radiolabelled probes in solution was followed by electrophoresis in 8-15% polyacrylamide gels (as appropriate to the size of the amplified sequence) and autoradiography.
  • Clone 470-20-1 was tested by genomic PCR, against human, E. coli , and yeast DNAs. No specific sequence was detected in negative control reactions, nor in any genomic DNA which was tested, and 10 5 copies of DNA/reaction resulted in a readily-detectable signal. This sensitivity (i.e., 10 5 /reaction) is adequate for detection of single-copy human sequences in reactions containing 1 ug total DNA, representing the DNA from approximately 1.5 x 10 5 cells. All clones discussed herein have tested negative for human DNA, Mystax DNA (mystax derived clones only) , E. coli , and yeast DNA using genomic PCR.
  • Serum or other cloning source or related source materials were directly tested by PCR using primers from selected cloned sequences.
  • putative N(ABCDE) viral particles DNA or RNA
  • PEG polyethylene glycol
  • PNF and certain other sera were pelleted by ultracentrifugation.
  • the pelleted materials were digested with proteinase k, followed by phenol/chloroform extraction and ethanol precipitation (Ausubel, et al . ) .
  • RNA For purification of RNA, the pelleted materials were dissolved in guanidinium thiocyanate and extracted by the acid guanidinium phenol technique (Chomczynski, et al . ) . Alternatively, total nucleic acids were prepared by proteinase k/sodium dodecyl sulfate (SDS) digestion and phenol/chloroform extraction, followed by alcohol precipitation.
  • SDS proteinase k/sodium dodecyl sulfate
  • 470-20-1 nucleic acid from the equivalent of 50 ul of PNF serum was used as the input template into each RT/PCR or PCR reaction.
  • Primers were designed based on the 470-20-1 sequence, as follows: 470-20-1-77F (SEQ ID NO:25) and 470-20-1-211R (SEQ ID NO:26).
  • Reverse transcription was performed using MMLV-RT (Gibco/BRL) and random hexamers (Promega, Madison, WI) by incubation at room temperature for approximately 10 minutes, 42°C for 15 minutes, and 99°C for 5 minutes, with rapid cooling to 4°C.
  • the synthesized cDNA was amplified directly, without purification, by PCR, in reactions containing 1.75 mM MgCl 2 , 1 uM each primer, 200 uM each dATP, dCTP, dGTP, and TTP, and 2.5 units Taq DNA polymerase ("AMPLITAQ", Perkin-Elmer, Norwalk, CT) per 100 ul reaction.
  • AMPLITAQ Perkin-Elmer, Norwalk, CT
  • Cycling was for at least one minute at 94°C, followed by 40-45 repetitions of denaturation (94°C for 15 seconds for 10 cycles; 92°C for 15 seconds for the succeeding cycles) , annealing (55°C for 30 seconds) , and extension (72°C for 30 seconds) , in the "GENEAMP SYSTEM 9600" thermal cycler (Perkin-Elmer) or comparable cycling conditions in other thermal cyclers (Perkin-Elmer; MJ Research, Watertown, MA) .
  • Positive controls consisted of previously amplified PCR product whose concentration was estimated using the Hoechst 33258 fluorescence assay, or purified plasmid DNA containing the DNA sequence of interest.
  • an aliquot of positive control DNA corresponding to approximately 10-100 copies/rxn. was spiked into reactions containing nucleic acids extracted from the cloning source specimen, as a control for the presence of inhibitors of DNA amplification reactions. Each separate extract was tested at least once in this manner. Specific products were detected by hybridization to a specific oligonucleotide probe 470-20-1-152R (SEQ ID NO:27), for confirmation of specificity.
  • Hybridization of 10 ul of PCR product was performed in solution in 20 ul reactions containing approximately 1 x 10 6 cpm of 32 P-labelled 470-20-1-152R. Specific hybrids were detected following electrophoretic separation from unhybridized oligo in polyacrylamide gels, and autoradiography.
  • the antigens D6, and D19 were screened using panels of sera derived Mystax both prior and subsequent to innoculation with the GB agent and/or PNF.
  • the antigens D6, D19, IA and 17A were screened using panels of human sera derived both from individuals suffering from hepatitis and uninfected controls.
  • the antigen sj26-pGEX-GLI-l was used as a control. This sample was determined to be at a concentration of 5 mg/ml using multiple assays, and was used as a standard for estimating the concentration of all other antigens tested.
  • Protein concentrations for all antigens were determined by a combination of determining the OD 280 nm of the protein fractions and by comparison to the protein standard described above. For all antigens 3 protein concentrations in the range of 0.5-3 ug/cm were fractionated using a 12.5% polyacrylimide gel and transferred onto nitrocellulose membrane. Typically 3- 7 cm of membrane containing each protein concentration would be produced. The membrane was then blocked in a solution of "BLOTTO" (150 mM NaCl, 20 mM tris-HCl pH 7.5, 1% normal goat serum, 1% Bovine serum albumin, 1% non-fat dry milk (w/v) and 0.02% sodium azide) at at least 1 hour. The membrane was then dried and cut into 1-2 mm strips.
  • BLOTTO 150 mM NaCl, 20 mM tris-HCl pH 7.5, 1% normal goat serum, 1% Bovine serum albumin, 1% non-fat dry milk (w/v) and 0.02% sodium azide
  • the strips containing different levels of blotted antigen were first rehydrated in TBS (150 mM NaCl; 20 mM Tris HCI, pH 7.5) then incubated overnight with test sera diluted in "BLOTTO" to which was added whole cell lysate of bacteria expressing non ⁇ recombinant pGEX-GLI at a dilution of 1/20 (v/v) .
  • TBS 150 mM NaCl
  • Tris HCI 20 mM Tris HCI, pH 7.5
  • test sera diluted in "BLOTTO" to which was added whole cell lysate of bacteria expressing non ⁇ recombinant pGEX-GLI at a dilution of 1/20 (v/v) .
  • one strip containing 3 ug/cm of nonrecombinant pGEX-GLI was also incubated with test sera.
  • TTBS TBS plus 0.2- 1.5% "TWEEN 20”
  • goatn anti-Human IgG conjugated to alkaline phosphatase diluted between 1/2000-1/7500 in "BLOTTO" for 1-2 hours at 25°C with agitation, at which point the strips were washed 4 times with TBS
  • Bound antibody was detected by incubating the strips in a substrate solution containing BCIP and NBT in pH 9.8 phosphate buffer. Color development was allowed to proceed for -15 minutes at which point color development was halted by 3 washed in distilled H 2 0. The concentration of antigen that gave the strongest signal with the least amount of background or non ⁇ specific reactivity was determined and employed for all subsequent assays.
  • nitrocellulose membranes Prior to testing with human or Mystax sera 18 X 13 cm nitrocellulose membranes were prepared using the optimum antigen concentrations determined as described above. The antigens were fractionated, and transferred to nitrocellulose as described above. The antigens were fractionated, and transferred to nitrocellulose as described above. Prior to large scale testing, four strips from each of the membranes to be employed were pre-tested by incubation with positive and control data as described above. All membranes used in large-scale testing had to demonstrate immunoreactivity to positive control sera prior to use. Assays with test sera were performed as is described above, with each sera to be tested being incubated with antigen containing pGEX-GLI strips, up to 80 sera were tested in any one assay. Test sera were derived from the following groups of individuals and/or experimental animals.
  • the antigens D6 and D19 were strongly reactive only with the screening serum Mystax 88. Neither antigen was reactive with pre-inoculate sera from any of the four mystax that were inoculated with GB agent.
  • Table 11 presents the results obtained from testing the antigens D6, D19, 17A and IA with human sera.
  • EXAMPLE 16 IMMUNOSCREENING OF A DEN cDNA LIBRARY A. Construction of DEN cDNA and DNA Libraries. A cDNA library was constructed, essentially as described in Example 2, using RNA isolated from DEN serum (Example 1) .
  • a DNA library was constructed, essentially as described in Example 3, using DNA isolated from DEN serum.
  • the DEN cDNA source (ATCC 75417) and DNA source (ATCC 75418) libraries were deposited at the American Type Culture Collection, 12301 Parklawn Dr., Rockville, MD 20852.
  • Clones DR25-1 and 2DR8 (i) tested positive for immunoreactivity in secondary plaque screening assays using DEN serum, (ii) was determined to be exogenous to human, mystax, and E. coli genomic DNA, (iii) was non- immunoreactive with normal human sera, and (iv) contained an open reading frame in frame with ⁇ - galactosidase.
  • sequences of clones DR25-1 and 2DR8 were compared with "GENBANK” and EMBL database sequences at both nucleic acid and amino acid levels.
  • the "GENBANK” search indicated that the sequences are unique as both nucleic acid and amino acid sequences.
  • the cDNA and DNA source libraries were deposited at Genelabs Technologies, Incorporated, 505 Penobscot Drive, Redwood City, CA 94063.
  • Clones whose designations begin with “468” and “472” were isolated from cDNA libraries made from My 670 serum. Clones whose names begin with “474" or “486” were isolated from cDNA and DNA libraries, respectively, made from My 620 serum. Clones whose names begin with "475" were isolated from DNA libraries made from My 670 serum.
  • AAACAGTTGA TGAATTTAGC AAATTCATAC TTTTTTTTCT GCGTGCCCCT ACCTATTACC 120
  • CTGCCTCTGC CTCTACCTCT TGAATATTCA TCTCCAAATT TCTATCAAAA TGACCAATGG 180

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Abstract

Polypeptide antigens which are immunoreactive with sera from individuals infected with non-A, non-B, non-C, non-D, non-E hepatitis are disclosed. Also disclosed are corresponding genomic-fragment clones containing polynucleotides encoding the open reading frame sequences for the antigenic polypeptides. Methods are presented for the isolation of sequences corresponding to the N-(ABCDE) hepatitis agents, including immunological and hybridization screening methods.

Description

NON-A/NON-B/NON-C/NON-D/NON-E HEPATITIS AGENTS AND MOLECULAR CLONING THEREOF
Field of Invention This invention relates methods for the isolation of sequences corresponding to N-(ABCDE) hepatitis agents, including immunological and hybridization screening meth¬ ods, and further, to DNA and cDNA libraries useful in such methods.
References
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Background of the Invention Viral hepatitis resulting from a virus other than hepatitis A virus (HAV) and hepatitis B virus (HBV) has been referred to as non-A, non-B hepatitis (NANBH) . One of these, known as enterically transmitted NANBH or ET- NANBH, is contracted predominantly in poor-sanitation areas where food and drinking water have been contaminated by fecal matter. The molecular cloning of the causative agent, referred to as the hepatitis E virus (HEV) , has recently been described (Reyes et al . (1990); Tarn et al . ) . A second NANB virus type, known as parenterally transmitted NANBH, or PT-NANBH, is transmitted by parenteral routes, typically by exposure to blood or blood products. Although the rate varies by locale, approximately 10% of transfusions cause PT-NANBH infection, and about half of these go on to a chronic disease state (Dienstag) . After anti-HCV testing, HCV seroconversion per unit transfused was decreased to less than 1% among heart surgery patients.
Human sera documented as having produced post-trans¬ fusion NANBH in human recipients have been used successfully to produce PT-NANBH infection in chimpanzees (Bradley) . RNA isolated from infected chimpanzee sera has been used to construct cDNA libraries in an expression vector for immunoscreening with chronic-state human PT- NANBH serum. This procedure identified a PT-NANBH specific cDNA clone and the viral sequence was then used as a probe to identify a set of overlapping fragments making up 7,300 contiguous basepairs of a PT-NANBH viral agent. The sequenced viral agent has been named the hepatitis C virus (HCV) (for example, the sequence of HCV is presented in EPO patent application 88310922.5, filed 11/18/88). The full-length sequence (~ 9,500 nt) of HCV is now available. Primate transmission studies conducted at the Centers for Disease Control (CDC; Phoenix, AZ, 1973-1975; 1978- 1983) originally provided substantial evidence for the existence of multiple agents of non-A, non-B (NANBH) : the primary agents of NANB are now recognized as being associated with infection by HCV and HEV (see above) . Later epidemiologic studies conducted at the CDC (Atlanta, GA, 1989-present) using both research (prototype) and commercial tests for anti-HCV antibody showed that approximately 20% of all community-acquired non-A, non-B hepatitis was also non-C. Further testing of these samples for the presence of HEV (Reyes, et al . , WO A 9115603 (Genelabs Inc.) 17 October 1991) have indicated that these cases of community-acquired non-A, non-B, non-C hepatitis were also non-E.
Liver biopsy specimens of Sentinel County patients obtained during the last five years (study of Drs. Miriam Alter and Kris Krawcynski) also showed that many bona fide cases of NANBH were also non-C hepatitis (serologically and RT-PCR (Kawasaki, et al . } Wang, et al . ) negative for all markers of HCV infection) developed subsequently into chronic hepatitis with presentation of chronic persistent hepatitis (CPH) or chronic active hepatitis (CAH) consistent with a viral infection.
Summary of the Invention
In a first aspect, the present invention includes a method of obtaining immunogenic polypeptides associated with non-A, non-B, non-C, non-D, non-E hepatitis agent (N- (ABCDE) ) infection. In this method, phage are prepared from a library selected from the group consisting of: MY 620 DNA source; MY 620 cDNA source; MY 670 DNA source; and MY 670 cDNA source. These libraries are deposited at Genelabs Technologies, Incorporated, 505 Penobscot Drive, Redwood City, CA 94063.
The phage are plated to form plaques and the phage plaques are screened for production of polypeptides immunoreactive with N-(ABCDE) serum. The serum used for screening the plaques can be from any N-(ABCDE) source, including human, mystax monkey or cynomolgus monkey serum. The invention further includes a method of obtaining non-A, non-B, non-C, non-D, non-E hepatitis agent (N-
(ABCDE) ) coding sequences associated with a region of a N- (ABCDE) genome. In this method phage are prepared that have insert sequences from a library selected from the group consisting of: MY 620 DNA source; MY 620 cDNA source; MY 670 DNA source; and MY 670 cDNA source. The phage are plated to form plaques. The plaques are then screened for production of polypeptides immunoreactive with N-(ABCDE) serum. Phage are isolated that produce polypeptides immunoreactive with N-(ABCDE) serum and the insert sequence is used to prepare hybridization probes. Phage are once again prepared from a library selected from the group consisting of MY 620 DNA source, MY 620 cDNA source, MY 670 DNA source, and MY 670 cDNA source, and plated to form plaques. The resulting plaques are screened using the hybridization probes. The hybridization probes can be selected from the sequences obtained by the method of the present invention.
The invention also includes the following libraries: MY 620 DNA source; MY 620 cDNA source; MY 670 DNA source; and MY 670 cDNA source.
In a second aspect, the present invention includes the isolation of polypeptide antigens that are immunoreactive with sera infected with a non-A, non-B, non-C, non-D, non-E hepatitis agent. Exemplary embodiments of the present invention include polypeptide antigens where an immunoreactive portion of the antigen is homologous to a polypeptide encoded by a sequence selected from the group consisting of: SEQ ID NO:11 through SEQ ID NO:16, SEQ ID NO:39 through SEQ ID NO:43, SEQ ID NO:74 through SEQ ID NO:104, and SEQ ID NO:106. In addition, a number of other antigenic peptides, useful in the practice of the present invention, are disclosed herein. Selected antigens of the present are encoded by the nucleic acid sequences presented as SEQ ID NO:11 through SEQ ID NO:16, SEQ ID NO:39 through SEQ ID NO:43, SEQ ID NO:74 through SEQ ID NO:104, and SEQ ID NO:106. These antigens may include heterologous protein sequences (i.e., they may be fused polypeptides) , such as sequences encoding /9-galactosidase or glutathione-S-transferase. The present invention also includes an expression system for expressing an antigenic polypeptide having an immunoreactive portion that is immunoreactive with sera infected with non-A, non-B, non-C, non-D, non-E hepatitis agent. The expression system typically includes a host capable of supporting expression of an open reading frame in a selected expression vector, where the selected expression vector includes an open reading frame of sequences encoding an immunoreactive portion of the polypeptide antigens described above. One useful expression vector is lambda gtll: other useful expression vectors are known in the art. Further, the present invention discloses a method of producing a polypeptide that is immunoreactive with N- (ABCDE) hepatitis sera. Typically, the polypeptide is produced by introducing a selected expression vector containing an open reading frame having sequences encoding the polypeptide antigen into a host capable of supporting expression of an open reading frame in the selected expression vector. The host cell is then cultured under conditions resulting in the expression of the open reading frame sequence. One useful expression vector is lambda gtll phage vector and the host is Escherichia coli .
A further embodiment of the present invention, is a cloning vector capable of expressing under suitable conditions an antigenic polypeptide having an immunoreactive portion that is immunoreactive with sera infected with non-A, non-B, non-C, non-D, non-E hepatitis agent. The antigen is homologous to one of the polypeptides identified by the methods of the present invention and includes those polypeptides encoded by a sequence selected from the group consisting of: SEQ ID NO:11 through SEQ ID NO:16, SEQ ID NO:39 through SEQ ID NO:43, SEQ ID NO:74 through SEQ ID NO:104, and SEQ ID NO:106.
One embodiment of the present invention includes a recombinantly produced N-(ABCDE) hepatitis agent poly¬ nucleotide that encodes a polypeptide which is immuno¬ reactive with N-(ABCDE) hepatitis infected sera, where said polynucleotide is selected from the group consisting of: SEQ ID NO:11 through SEQ ID NO:16, SEQ ID NO:39 through SEQ ID NO:43, SEQ ID NO:74 through SEQ ID NO:104, and SEQ ID NO:106. Alternatively, the polynucleotide can correspond to other sequences obtained by the method of the present invention.
Another embodiment of the present invention includes a recombinantly produced N-(ABCDE) hepatitis agent polypeptide which is homologous to a polypeptide encoded by a sequence selected from the group consisting of: SEQ ID NO:11 through SEQ ID NO:16, SEQ ID NO:39 through SEQ ID NO:43, SEQ ID NO:74 through SEQ ID NO:104, and SEQ ID NO:106. Alternately, the recombinant polypeptide can be homologous to other polypeptides antigens obtained by the method of the present invention. Also included in the invention is a diagnostic kit for use in screening serum containing antibodies specific against N-(ABCDE) hepatitis infection. The kit includes a recombinant N-(ABCDE) hepatitis polypeptide antigen. Exemplary of such polypeptide antigens are polypeptides where an immunoreactive portion of said antigen is homologous to a polypeptide encoded by a sequence selected from the group consisting of: SEQ ID NO:11 through SEQ ID NO:16, SEQ ID NO:39 through SEQ ID NO:43, SEQ ID NO:74 through SEQ ID NO:104, and SEQ ID NO:106. The kit also includes means for detecting the binding of said antibodies to the antigen. One means for detecting the binding of antibodies to the antigen includes a solid support to which the N-(ABCDE) hepatitis polypeptide antigen is attached and a reporter-labelled anti-human antibody, where binding of serum antibodies to the antigen can be detected by binding of the reporter-labelled anti- body to said serum antibodies. The can be used in a method of detecting N-(ABCDE) hepatitis agent infection in a primate. In this method the serum from a N-(ABCDE) hepatitis test primate is reacted with a recombinant N- (ABCDE) hepatitis polypeptide antigen and the antigen is examined for the presence of bound antibody.
The present invention also includes a diagnostic kit for use in screening samples containing N-(ABCDE) hepatitis agent nucleic acids. This kit contains primers having sequences specific to N-(ABCDE) hepatitis agents. For example, such sequences can be selected from the group consisting of: SEQ ID NO:11 through SEQ ID NO:16, SEQ ID NO:39 through SEQ ID NO:43, SEQ ID NO:74 through SEQ ID NO:104, and SEQ ID NO:106. The primer may contain reporter moieties for hybridization detection. These primers may be used in a method of detecting N-(ABCDE) hepatitis agent nucleic acid in a primate. In the method a nucleic acid sample is obtained from the primate subject. The sample is combined with at least one primer containing sequences specific to N-(ABCDE) hepatitis agents. The presence of N-(ABCDE) hepatitis agent nucleic acid/primer complexes, formed by hybridization of the N- (ABCDE) hepatitis nucleic acid with primer, is then detected. In addition to hybridization detection using labelled primers, sets of primers can be used to identify target N-(ABCDE) hepatitis agent nucleic acid in a sample by use of polymerase chain reaction amplification. Further, a capture moiety may be included in a primer to allow capture of target sequences to which it binds (e.g., biotin incorporated, avidin capture) . These and other objects and features of the invention will be more fully appreciated when the following detailed description of the invention is read in conjunction with the accompanying drawings.
Brief Description of the Figures Figure 1: the use of PNF 2161 plasma in primate transmission studies is diagrammed.
Figure 2: the GB sera use in cross challenge to mystax infected with passaged sera derived from PNF 2161 is diagrammed. Figure 3: illustrates the results of a Western blot analysis of the membrane containing crude lysates containing the pGEX-GLI-D6 and pGEX-GLI-D19 encoded antigens of the present invention.
Figures 4A and 4B: illustrate the results of Western blots of the partially purified Dl, D6 and D19 fusion proteins using mystax (My 88) serum (Figure 4A) and N-
(ABCDE) human (Ruf) serum (Figure 4B) .
Figures 5A, 5B and 5C: illustrate the result of
Western blots of crude lysates of JFA-17A and JFA-1A fusion proteins. Figure 5A is a photograph of a coomaisse blue stained polyacrylamide gel on which the samples were separated. Figure 5B shows a Western blot analysis of the
Figure 5A polyacrylamide gel transferred to a membrane and probed with JFA serum. In Figure 5B the arrows indicate the locations of the SJ26-17A and SJ26-1A fusion proteins, first and second panels, respectively. Figure 5C shows the Western blot analysis of Figure 5A polyacrylamide gel transferred to a membrane and probed with normal human serum. Figure 6: schematically illustrates the overlap between clone 17A and WT54.
Figure 7: schematically represents the steps taken in the polymerase chain reaction linking experiments for clones JFA-17A and WT54. Detailed Description of the Invention
1. Definitions
The terms defined below have the following meaning herein: 1. "nonA/nonB/nonC/nonD/nonE hepatitis viral agent N-(ABCDE)" means a virus, virus type, or virus class which (i) is transmissible in primates (e . g. , mystax, cynomolgus and marmoset monkeys, chimpanzees, or humans) , and (ii) is serologically distinct from hepatitis A virus (HAV) , hepatitis B virus (HBV) , hepatitis C virus (HCV) , hepatitis D virus, and hepatitis E (HEV) .
2. Two nucleic acid fragments are considered to have "homologous hybridizable" sequences if they are capable of hybridizing to one another (i) under typical hybridization and wash conditions, as described, for example, in Maniatis, et al . , pages 320-328, and 382-389, or (ii) using reduced stringency wash conditions that allow at most about 25-30% basepair mismatches, for example: 2 x SSC, 0.1% SDS, room temperature twice, 30 minutes each; then 2 x SSC, 0.1% SDS, 37°C once, 30 minutes; then 2 x
SSC, room temperature twice, 10 minutes each. Preferably, homologous nucleic acid strands contain 15-25% basepair mismatches, even more preferably 5-15% basepair mismatches. These degrees of homology can be selected by using wash conditions of appropriate stringency for identification of clones from gene libraries (or other sources of genetic material) , as is well known in the art.
3. Two amino acid sequences or two nucleotide sequences (in an alternative definition for homology between two nucleotide sequences) are considered homologous (as this term is preferably used in this specification) if they have an alignment score of >5 (in standard deviation units) using the program ALIGN with the mutation gap matrix and a gap penalty of 6 or greater (Dayhoff) . The two sequences (or parts thereof, preferably at least 35 amino acids in length) are homologous if their amino acids alignments are greater than or equal to 40%, preferably 60% and more preferably 80% using the ALIGN program mentioned above.
4. A DNA or cDNA fragment is "derived from" N- (ABCDE) viral agents if it has the same or substantially the same basepair sequence as a cloned region of a N- (ABCDE) viral agent genome.
5. A protein is "derived from" N-(ABCDE) viral agents if it is encoded by an open reading frame of a DNA or RNA fragment derived from a N-(ABCDE) viral agent or displays homology as noted under Example 3 above.
6. In the context of the present invention, the phrase "nucleic acid sequences," when referring to sequences which encode a protein, polypeptide, or peptide, is meant to include degenerative nucleic acid sequences which encode homologous .protein, polypeptide or peptide sequences as well as the disclosed sequence.
7. In two or more known peptide sequences which are more than about 70% homologous in amino acid sequence, a third amino acid sequence will be internally consistent with the known sequences if each amino acid in the third sequence is identical to at least one of amino acids in the known sequences.
II. N-(ABCDE) Sera. Originally, infectivity studies of a putative viral agent of human origin, termed "GB," relied on the use of serum derived from acutely infected tamarins (several species) and marmosets (Callithrix jacchuε) (Deinhardt, et al . , 1967). Studies at the Centers for Disease Control (CDC; Phoenix AZ) involved the use of eleventh passage (Pll) "GB" serum which was inoculated intravenously into naive tamarins. All tamarins inoculated with the material developed relatively severe, short-incubation-period hepatitis suggestive of passage or adaptation of this putative human agent to the genus of tamarins or that the agent itself was of non-human (primate) origin. The eleventh passage "GB" agent is available from the American Type Culture Collection, 12301 Parklawn Dr. , Rockville MD 20852, as VR-806. This is Pll of the original acute-phase human serum stored in liquid nitrogen.
Two other passages of the GB agent, obtained from Dr. Purcell, National Institutes of Health (NIH) , Bethesda, MD were also used. S. mystax (MY 620, MY 661, MY 663, and MY 670) were inoculated intravenously with GB sera by Dr. Purcell. S . labiatus (539B, 500, and 41B; Karayinnis, et al . , 1989) were inoculated intravenously with GB sera by Professor Thomas and Dr. Karayinnis. All animals developed symptoms of non C hepatitis.
The arrival of a serologic test for anti-HCV and the development of an RT-PCR (Kawasaki, et al . ; Wang, et al . ) for HCV-RNA allowed the identification of several cases of both post-transfusion and sporadic non-HCV hepatitis. Human hepatitis case, PNF-2161, had been followed for at least 60 months, with blood collection every three months during the period following the acute-phase of disease.
Although initially diagnosed as having chronic non-C hepatitis, this individual (PNF 2161) was subsequently discovered to be co-infected with HCV: (i) RT-PCR for the 5,UTR of HCV-RNA showed that HCV was indeed present in• this plasma; (ii) further antibody testing showed the presence of anti-HCV antibodies (see Example 1, Table 1) . Inoculation of sera and plasma from case PNF-2161 into two chimpanzees, however, showed no evidence of disease due to HCV, except for the development of anti-capsid (C22) antibody in one animal. The other animal showed no ALT elevation or serologic evidence of infection with HCV. The results of inoculations in these two animals indicate that the HCV-variant present in the PNF 2161 serum was highly atypical, in that, in numerous animal studies even low titer inocula containing HCV elicited reproducible hepatitis infection in chimpanzees. These findings indicated the presence of another etiologic agent of PT- 14
NANBH, other than HCV, in the chronic-phase plasma of case PNF-2161.
In view of the inability of the PNF 2161 plasma to transmit HCV infection to chimpanzees and the possible susceptibility of Sanguinis mystax (mystax) and cynomologus macaques (cynos) to infection with the GB hepatitis agent, mystax and cynos monkeys were challenged with PNF 2161 plasma. Intravenous inoculation of cynos and mystax with PNF 2161 resulted in obvious elevations of liver enzymes, including serum ALT activity, and histologic correlates of a viral infection (Figure 1) . The latter findings were most obvious in the liver of MY 131, whose liver was then obtained at or near the peak of ALT activity. Previous studies have indicated that HCV will not infect mystax monkeys. Furthermore, selected animals from these studies showed no serologically markers of HCV infection. To strengthen the argument that PNF 2161 contains an infectious and transmissible agent of presumed viral origin, subpassage studies were performed in both mystax and cynos.
Figure 1 illustrates serial passage of non-HCV hepatitis in both species of primate and further illustrates that the etiologic agent is chloroform- resistant. In Figure 1, PNF 2161 is the original non-C serum that was used to inoculate two chimpanzees (CH1323, with 1 ml. of pooled sera, and CH1356, with 10 ml. of plasma) , tamarins (Sanguinis mystax; MY, with 1 ml. of plasma) and cynos (Cynomolgus macaques; CY, with 1 ml. of plasma) . These animals were then monitored for elevated ALT or SICD values (Bradley, et al . , 1987, op. cit . ) : elevated values in the animal are indicated by an asterisk. As noted above, MY 131 had very significant elevated ALT values. The liver of MY131 was obtained at or near the peak of ALT activity. A liver homogenate was prepared and diluted. Ten percent and 1% liver solutions were inoculated into S . mystax primates. A 1% liver solution was inoculated into cynos primates. For each inoculation the liver was either treated or not treated with chloroform prior to injection. As above, ALT and SICD values were determined for each animal. Chronic-phase plasma from two other bona fide cases of non-HCV hepatitis were obtained from Sentinel County Study patients JFA-2179 and DEN-2492 (arranged in consultation/collaboration with Dr. Miriam Alter, P.I. of this Sentinel County Study) . Ten-ml aliquots of each of these plasma were intravenously inoculated into two separate chimpanzees. As of approximately 100 days post- inoculation (4/13/92) neither chimpanzee had shown biochemical (ALT) evidence of liver disease or serologic evidence of infection with HCV. Aliquots of these same plasma were used in transmission studies with S . mystax. In view of the fact that the GB hepatitis agents and the chloroform-resistant agent recovered from the plasma of case PNF-2161 could infect S . mystax, a cross-challenge study was conducted. In Figure 2, the GB sera had been passed in marmosets (Callithrix jacchus) ; the Pll sera was inoculated intravenously into naive S . mystax (MY 88 and MY 190) . The Pll sera was also injected into 3 selected animals, MY 73, MY 136, and MY 242. These S . Mystax had been infected first with the PNF-2161 derived agent, they were then intravenously inoculated with serum from a marmoset (passage #11; 1.0 ml aliquots of ATCC VR-806 diluted 1:10 in PBS). All three tamarins MY 73, MY 136, and MY 242 developed a clear second bout of non-HCV hepatitis, indicating that either the etiologic agents were different, or that immunoprotection against reinfection by the same agent was absent (that is, no neutralizing antibodies were present in the convalescent sera of tamarins infected with the PNF-2161 agent) . Tamarins MY 88 and MY 190 also developed non-C hepatitis. In addition to the above described sera a number of human N-(ABCDE) sera have also been identified including SCH (Example 1) . Animal transmission and cross-challenge studies of this type indicate the basic methodology used in the identification of new blood-born pathogenic viruses.
III. Isolation of N-fABCDE) Hepatitis Agent Sequences.
As one approach toward identifying clones containing N-(ABCDE) hepatitis sequences, cDNA and DNA libraries were prepared from infected sera in the expression vector lambda gtll (Examples 2, 3 and 16) . cDNA and DNA sequences were then selected for the expression of peptides which are immunoreactive with N-(ABCDE) hepatitis infected sera. First round screening was typically performed using the same sera that was used to generate the phage library, or, alternatively, a closely related sera. It is also possible to screen with other suspected N-(ABCDE) sera.
Recombinant proteins identified by this approach provide candidates for peptides which can serve as substrates in diagnostic tests. Further, the nucleic acid coding sequences identified by this approach serve as useful hybridization probes for the identification of further N-(ABCDE) hepatitis coding sequences.
The sera described above were used to generate cDNA and DNA libraries in lambda gtll (Examples 2, 3 and 16). In the method illustrated in Example 2, infected serum was precipitated in 8% PEG without dilution, and the libraries were generated from the resulting pelleted virus. Sera from infected human sources were treated in the same fashion.
As an advantageous alternative to PEG precipitation, ultracentrifugation can be used to pellet particulate agents from infected sera or other biological specimens. To isolate viral particles from which nucleic acids could be extracted, serum, ranging up to 2 ml, was diluted to approximately 10 ml with PBS and was centrifuged for a minimum of 2 hours at 40,000 rpm (approximately 110,000 x g) in the Ti70.1 rotor (Beckman Instruments, Fullerton, CA) at 4°C. The supernatant was the aspirated and the pellet extracted by standard nucleic acid extraction techniques. cDNA libraries were generated using random primers in reverse transcription reactions with RNA extracted from pelleted sera as starting material. DNA libraries were generated by proteinase K treatment and SDS lysis of pelleted sera, followed by the addition of Klenow fragment of DNA polymerase and random primers to the nucleic acid. The resulting molecules, cDNA or DNA, were ligated to SISPA (Reyes, et al . , (1991)) linker primers and expanded in a non-selective manner, and then cloned into a suitable vector, for example, lambda gtll, for expression and screening of peptide antigens, and the lambda gtlO vector, for hybridization screening.
Lambda gtll is a particularly useful expression vector which contains a unique EcoRI insertion site 53 base pairs upstream of the translation termination codon of the ?-galactosidase gene. Thus, an inserted sequence is expressed as a ?-galactosidase fusion protein which contains the N-terminal portion of the ?-galactosidase gene product, the heterologous peptide, and optionally the C-terminal region of the ?-galactosidase peptide (the C— terminal portion being expressed when the heterologous peptide coding sequence does not contain a translation termination codon) . This vector also produces a temperature-sensitive repressor (cI857) which causes viral lysogeny at permissive temperatures, e.g., 32°C, and leads to viral lysis at elevated temperatures, e.g., 42°C.
Advantages of this vector include: (1) highly efficient recombinant clone generation, (2) ability to select lysogenized host cells on the basis of host-cell growth at permissive, but not non-permissive, temperatures, and (3) high levels of recombinant fusion protein production. Further, since phage containing a heterologous insert produces an inactive ?-galactosidase enzyme, phage with inserts are typically identified using a yff-galactosidase colored-substrate reaction.
Examples 2, 3 and 16 describe the preparation of a cDNA and DNA library for each of the following N-(ABCDE) hepatitis sera: MY 131, MY 190, MY 620, MY 670, PNF 2161, JFA, SCH and DEN. These libraries were immunoscreened using N-(ABCDE) hepatitis positive human or mystax sera (Examples 4, and 7-11). A number of lambda gtll clones were identified which were immunoreactive with at least one of the sera. Immunopositive clones were plaque-purified and their immunoreactivity retested. Also, the immunoreactivity of the clones with pre-inoculu mystax and/or normal human sera was also tested.
These clones were also examined for the "exogenous" nature of the cloned insert sequence. This basic test establishes that the cloned fragment does not represent a portion of the human or other known (e.g. bacterial) genomes. The clone inserts were isolated by EcoRI digestion following polymerase chain reaction amplification. The inserts were purified then radiolabelled and used as hybridization probes against membrane bound normal human DNA, normal mystax DNA and bacterial DNA (control DNAs) (Example 6) .
Described below are a number of clones that were (i) immunoreactive with the N-(ABCDE) hepatitis test sera, (ii) exogenous to human, normal, and bacterial genomes, (iii) not immunologically reactive with pre-immune mystax and/or normal human sera, and (iv) had unique nucleic acid sequences when compared with one another. The latter may indicate the isolation of multiple viruses or the isolation of different immunogenic regions from the same genome. The sequences of these clones are presented in the Sequence Listing. Furthermore, the sequences of the cloned inserts, when searched against the "GENBANK" sequence library, were not found to have significant homology to any known sequences, including those from known hepatitis virus sequences. Other characteristics of a number of the sequenced clones follow here. The D19 clone (MY 190 DNA source library) also was shown to be exogenous to normal human, tamarin and bacterial DNA. The clone has a large open reading frame (104 bp) , in frame with the /3-galactosidase gene of the lambda gtll vector (SEQ ID NO:17). The antigen encoded by D19 was shown to be immunoreactive with 2/9 infected mystax sera.
Clone 17A (JFA DNA source) was shown to be exogenous to normal human and bacterial DNA. The clone has a large open reading frame (590 bp) , in frame with the β- galactosidase gene of the lambda gtll vector (SEQ ID NO:36). The 17A antigen has been expressed as a GST fusion protein and tested immunopositive with JFA serum. Clone IA (JFA DNA source) was shown to be exogenous to normal human and bacterial DNA. The clone has a large open reading frame (467 bp) , in frame with the β- galactosidase gene of the lambda gtll vector (SEQ ID NO:37). The antigen encoded by clone IA has been expressed as a GST fusion protein and tested immunopositive with JFA serum and negative with normal human sera by Western blot analysis.
Some clones have multiple insert sequences as indicated by internal SISPA primers (Example 2) : for example, JFA clone 4B11 (SEQ ID NO:38), D12-3 (SEQ ID N0:46), D31-2 (SEQ ID NO:48) and D76 (SEQ ID NO:50) have 3 inserts; and R27 has four inserts (SEQ ID NO:33). In such cases the insert can be (i) fractionated into discrete sequences by restriction enzyme digestion with EcoRI or NotI , or (ii) portions of the insert separately PCR amplified by sequence specific primers. Each resulting individual region of the cloned sequence can be subcloned into, for example, lambda gtll or pGEX-GLI and immunoscreened as described above. This allows identification of specific regions responsible for the immunoreactivity (see Epitope Mapping, below) .
Clone 470-20-1 (PNF2161 cDNA source) was isolated by immunoscreening with the same cloning source. The clone was not reactive with normal human sera. The clone has a large open reading frame (203 base pairs; SEQ ID NO:106), in-frame with the ,9-galactosidase gene of the lamdba gtll vector. The clone is exogenous by genomic DNA hybridization analysis and genomic PCR analysis, using human, yeast and E. coli genomic DNAs. The sequence was present in PNF2161 serum as determined by RT-PCR. The sequence was also detected in sucrose density gradient fractions at densities consistent with the sequence banding in association with a virus-like particle. Further sequences (PNF2161-470-20-1 EXT1; SEQ ID
NO:104) adjacent to clone 470-20-1 were obtained by anchor polymerase chain reaction using primers from clone 470-20- 1 (Example 7) .
IV. Further Characterization of N-fABCDE) Hepatitis Recombinant Antigens.
A. Screening Recombinant Libraries.
Further candidate N-(ABCDE) hepatitis antigens can be obtained from the libraries of the present invention using the screening methods described above. The libraries described above have been deposited with the American Type Culture Collection, 12301 Parklawn Dr., Rockville MD 20852 and have been assigned the following designations: MY 131 cDNA source, ATCC 75273; MY 131 DNA source, ATCC 75270; MY 190 DNA source, ATCC 75284; MY 190 cDNA source; JFA cDNA source, ATCC 75272; JFA DNA source, ATCC 75271; SCH cDNA source, ATCC 75283; SCH DNA source, ATCC 75282; PNF 2161 CDNA source, ATCC 75268; PNF 2161 DNA source, ATCC 75269; DEN cDNA source, ATCC 75417; and DEN DNA source, ATCC 75418. The MY 620 cDNA source, MY 620 DNA source, MY 670 cDNA source, MY 670 DNA source and subtracted SCH DNA source libraries are deposited at Genelabs Technologies, Incorporated, 505 Penobscot Drive, Redwood City, CA 94063.
In addition to the recombinant libraries generated above, other recombinant libraries from N-(ABCDE) hepatitis sera can likewise be generated and screened as described herein.
B. Epitope Mapping, Cross Hybridization and Isolation of Genomic Sequences. The antigen-encoding DNA fragment can be subcloned. The subcloned insert can then be fragmented by partial DNase I digestion to generate random fragments or by specific restriction endonuclease digestion to produce specific subfragments. The resulting DNA fragments can be inserted into the lambda. gtll vector and subjected to immunoscreening in order to provide an epitope map of the cloned insert.
In addition, the DNA fragments can be employed as probes in hybridization experiments to identify overlapping N-(ABCDE) hepatitis sequences, and these in turn can be further used as probes to identify a set of contiguous clones. The generation of sets of contiguous clones allows the elucidation of the sequence of a given N-(ABCDE) hepatitis agent's genome. The following illustrates several approaches for the use of cloned insert-derived DNA to identify clones carrying other N-(ABCDE) hepatitis sequences. The insert of clone JFA-17A was isolated and used as a hybridization probe against the individual cDNA or DNA libraries established in a lambda vector (Example 10, Sections 10D and 10E) . Using the clone JFA-17A probe an overlapping clone has been isolated from a library synthesized from the JFA SISPA DNA source: this clone was designated WT54. The insert of the overlapping clone can be isolated by restriction enzyme digestion of the clone WT54, followed by electrophoretic fractionation and electroelu- tion. The isolated insert is then treated with DNase I to generate random fragments and the resulting digested frag¬ ments are inserted into lambda gtll phage vectors for immunoscreening to yield other immunoreactive regions.
Any of the above-described clone inserts can be used in a similar manner to probe the cDNA and DNA libraries generated in a vector, such as lambda gtlO or "LAMBDA ZAP II". Entire inserts of specific subfragments of any clone may be isolated by polymerase chain reaction or after cleavage with restriction endonucleases. These fragments can be used as radiolabelled probes against any selected library. In particular, the 5' and 3' terminal sequences of the clone inserts are useful as probes to identify additional clones. In addition, the clone inserts can be used to screen other libraries, for example, the D19 clone insert can be used to screen libraries generated from other human N-(ABCDE) hepatitis sera (such as SCH source libraries) .
Further, the sequences provided by the 5' end of cloned inserts are useful as sequence specific primers in first-strand cDNA or DNA synthesis reactions (Maniatis et al . ; Scharf et al . ) . For example, specifically primed MY 190 cDNA and DNA libraries can be prepared by using a D19 specific primer on My 190 nucleic acids as a template. The second-strand of the new cDNA is synthesized using RNase H and DNA polymerase I. The above procedures identify or produce DNA/cDNA molecules corresponding to nucleic acid regions that are 5' adjacent to the known clone insert sequences. These newly isolated sequences can in turn be used to identify further flanking se- quences, and so on, to identify the sequences composing the entire genome for a given N-(ABCDE) hepatitis agent. As described above, after new N-(ABCDE) hepatitis sequences are isolated, the polynucleotides can be cloned and immunoscreened to identify specific sequences encoding N-(ABCDE) hepatitis antigens.
Such extension clones, containing further sequences of interest, have been obtained for clone PNF 470-20-1 (SEQ ID NO:106): for example, extension clone (PNF2161- 470-20-1 EXT1, SEQ ID NO:104; Example 7).
C. Preparation of Antigenic Polypeptides and Antibodies.
The recombinant peptides of the present invention can be purified by standard protein purification procedures which may include differential precipitation, molecular sieve chromatography, ion-exchange chromatography, isoelectric focusing, gel electrophoresis and affinity chromatography. In the case of a fusion protein, such as the /9-galactosidase fusion proteins described above, the fused protein can be isolated readily by affinity chromatography, by passing cell lysis material over a solid support having surface-bound anti-yff-galactosidase antibody. For example, purification of a yff-galactosi- dase/fusion protein, derived from D19 coding sequences, by affinity chromatography is described in Example 12.
Fusion proteins containing the polypeptide antigens of the present invention fused with the glutathione-S- transferase (Sj26) protein have also been expressed using the pGEX-GLI vector system in E. coli JM101 cells (Examples 9D, 9E, and 12) . The fused Sj26 protein can be isolated readily by glutathione substrate affinity chromatography (Smith, et al . ) . Expression and partial purification of the D6, and D19 as GST (sj26) fusion proteins is described in Examples 9C, 9D and 12, and is applicable to any of the other soluble, induced antigens coded by sequences described by the present invention. Insoluble GST (sj26) fusion proteins (such as, the GST fusions containing the JFA-1A and JFA-17A antigens) have been purified by preparative gel electrophoresis.
Also included in the invention is an expression vector, such as the lambda gtll or pGEX vectors described above, containing N-(ABCDE) antigen coding sequences and expression control elements which allow expression of the coding regions in a suitable host. The control elements generally include a promoter, translation initiation codon, and translation and transcription termination se¬ quences, and an insertion site for introducing the insert into the vector.
The DNA encoding the desired antigenic polypeptide can be cloned into any number of commercially available vectors to generate expression of the polypeptide in the appropriate host system. These systems include: baculovirus expression (Reilly, et al . ; Beames, et al . ; Pharmigen; Clontech) , expression in bacteria (Ausubel, et al . ; Clontech), expression in yeast (Goeddel; Guthrie and Fink) , expression in mammalian cells (Clontech; Gibco- BRL) . These recombinant polypeptide antigens can be expressed as fusion proteins or as native proteins. A number of features can be engineered into the expression vectors, such as leader sequences which promote the secretion of the expressed sequences into culture medium. The recombinantly produced N-(ABCDE) hepatitis polypeptide antigens are typically isolated from lysed cells or culture media. Purification can be carried out by methods known in the art including salt fractionation, ion exchange chromatography, and affinity chromatography. Immunoaffinity chromatography can be employed using antibodies generated based on the N-(ABCDE) hepatitis antigens identified by the methods of the present invention.
The N-(ABCDE) hepatitis polypeptide antigens may also be isolated from N-(ABCDE) hepatitis agent particles (see below) .
Antigenic regions of polypeptides are generally relatively small, typically 7 to 10 amino acids in length. Smaller fragments have been identified as antigenic regions. N-(ABCDE) hepatitis polypeptide antigens are identified as described above. The resulting DNA coding regions can be expressed recombinantly either as fusion proteins or isolated polypeptides. In addition, some amino acid sequences can be conveniently chemically synthesized (Applied Biosystems, Foster City CA) . Antigens obtained by any of these methods may be directly used for the generation of antibodies or they may be coupled to appropriate carrier molecules. Many such carriers are known in the art and are commercially available (e . g. , Pierce, Rockford IL) .
In another aspect, the invention includes specific antibodies directed against the polypeptide antigens of the present invention. Typically, to prepare antibodies, a host animal, such as a rabbit, is immunized with the purified antigen or fused protein antigen. Hybrid, or fused, proteins may be generated using a variety of coding sequence derived from other proteins, such as β-galac- tosidase or glutathione-S-transferase. The host serum or plasma is collected following an appropriate time inter¬ val, and this serum is tested for antibodies specific against the antigen. Example 13 describes the production of rabbit serum antibodies which are specific against the D19 antigens in the SJ26/D19 hybrid protein. These techniques are equally applicable to the other antigens of the present invention.
The gamma globulin fraction or the IgG antibodies of immunized animals can be obtained, for example, by use of saturated ammonium sulfate precipitation or DEAE Sephadex chromatography, or other techniques known to those skilled in the art for producing polyclonal antibodies.
Alternatively, purified antigen or fused antigen pro¬ tein may be used for producing monoclonal antibodies. Here the spleen or lymphocytes from an immunized animal are removed and immortalized or used to prepare hybridomas by methods known to those skilled in the art. To produce a human-human hybridoma, a human lymphocyte donor is selected. A donor known to be infected with a N-(ABCDE) hepatitis agent may serve as a suitable lymphocyte donor. Lymphocytes can be isolated from a peripheral blood sample. Epstein-Barr virus (EBV) can be used to immortalize human lymphocytes or a human fusion partner can be used to produce human-human hybridomas. Primary in vitro sensitization with viral specific polypeptides can also be used in the generation of human monoclonal antibodies.
Antibodies secreted by the immortalized cells are screened to determine the clones that secrete antibodies of the desired specificity, for example, by using the ELISA or Western blot method (Ausubel et al . ) .
D. ELISA and Protein Blot Screening. When N-(ABCDE) antigens are identified, typically through plaque immunoscreening as described above, the antigens can be expressed and purified. The antigens can then be screened rapidly against a large number of suspected N-(ABCDE) hepatitis sera using alternative immunoassays, such as, ELISAs or Protein Blot Assays (i . e . , Westerns) employing the isolated antigen peptide. The antigen polypeptides fusion can be isolated as described above, usually by affinity chromatography to the fusion partner such as jS-galactosidase or glutathione-S- transferase. Alternatively, the antigen itself can be purified using antibodies generated against it (see below) . A general ELISA assay format is presented in the Materials and Methods section below. Harlow, et al . , describe a number of useful techniques for immunoassays and antibody/antigen screening.
The purified antigen polypeptide or fusion polypeptide containing the antigen of interest, is attached to a solid support, for example, a multiwell polystyrene plate. Sera to be tested are diluted and added to the wells. After a period of time sufficient for the binding of antibodies to the bound antigens, the sera are washed out of the wells. A labelled reporter antibody is added to each well along with an appropriate substrate: wells containing antibodies bound to the purified antigen polypeptide or fusion polypeptide containing the antigen are detected by a positive signal.
A typical format for protein blot analysis using the polypeptide antigens of the present invention is presented in Example 15. General protein blotting methods are described by Ausubel, et al . In Example 15, fusion proteins containing the antigens JFA-1A, JFA-17A, D6 and D19 were used to screen a number of sera samples. The results presented in Example 15 demonstrate that several different source N(ABCDE) Hepatitis sera are immunoreactive with these polypeptide antigens.
These results demonstrate that the polypeptide antigens of the present invention can, by these methods, be rapidly screened against panels of N-(ABCDE) hepatitis serum samples.
E. Cell Culture Systems, Animal Models and Isolation of N-(ABCDE) Hepatitis Agents.
N-(ABCDE) hepatitis agents may be propagated in the animal model systems described above. The N-(ABCDE) hepatitis agents described in the present specification have the advantage of being capable of infecting marmoset and cynos monkeys. This provides a convenient and accessible animal model as well as an animal model that discriminates against the propagation of HCV.
Alternatively, primary hepatocytes obtained from infected animals (chimpanzees, baboons, monkeys, or humans) can be cultured in vitro . A serum-free medium, supplemented with growth factors and hormones, has been described which permits the long-term maintenance of differentiated primate hepatocytes (Lanford, et al . ; Jacob, et al . , 1989, 1990, 1991). In addition to primary hepatocyte cultures, immortalized cultures of infected cells may also be generated. For example, primary liver cultures may be fused to a variety of cells (like HepG2) to provide stable immortalized cell lines. Primary hepatocyte cell cultures may also be immortalized by introduction of oncogenes or genes causing a transformed phenotype. Such oncogenes or genes can be derived from a number of sources known in the art including SV40, human cellular oncogenes and Epstein Barr Virus. Further, the un-infected primary hepatocytes may be infected by exposing the cells in culture to the N-(ABCDE) hepatitis agents either as partially purified particle preparations (prepared, for example, from infected sera by differential centrifugation and/or molecular sieving) or in infectious sera. These infected cells can then be propagated and the virus passaged by methods known in the art.
In addition to expression of the N-(ABCDE) hepatitis infectious agents, regions of the N-(ABCDE) hepatitis agent genomic information can be introduced by recombinant means into the hepatocyte cells. Such recombinant manipulations allow the individual expression of individual components of the N-(ABCDE) hepatitis agent genomes. RNA samples can be prepared from infected tissue or, in particular, from infected cell cultures. The RNA samples can be fractionated on gels and transferred to membranes for hybridization analysis using probes derived from the cloned N-(ABCDE) hepatitis sequences. N-(ABCDE) hepatitis particles may be isolated from infected sera, infected tissue, the above-described cell culture media, or the cultured infected cells by methods known in the art. Such methods include techniques based on size fractionation (i.e., density centrifugation, precipitation, ultracentrifugation) , using anionic and/or cationic exchange materials, separation on the basis of hydrophilic properties, and affinity chromatography. During the isolation procedure the N-(ABCDE) hepatitis agents can be identified using the anti-N-(ABCDE) hepatitis antibodies of the present invention or by using hybridization probes based on identified N-(ABCDE) hepatitis agent nucleic acid sequences. Antibodies directed against the N-(ABCDE) hepatitis agents can be used in purification of N-(ABCDE) hepatitis particles through immunoaffinity chromatography (Harlow, et al . ; Pierce). Antibodies directed against N-(ABCDE) hepatitis polypeptides or fusion polypeptides (such as
D19) are fixed to solid supports in such a manner that the antibodies maintain their immunoselectivity. To accomplish such attachment of antibodies to solid support bifunctional coupling agents (Pierce; Pharmacia) containing spacer groups are frequently used to retain accessibility of the antigen binding site of the antibody.
N-(ABCDE) hepatitis particles can be further characterized by standard procedures including immunofluorescence microscopy, electron microscopy, Western blot analysis of proteins composing the particles, infection studies in animal and/or cell systems utilizing the partially purified particles, and sedimentation characteristics.
The N-(ABCDE) hepatitis particles can be disrupted to obtain N-(ABCDE) hepatitis genomes. Disruption of the particles can be achieved by, for example, treatment with detergents in the presence of chelating agents. The genomic nucleic acid can then be further characterized. Characterization may include analysis of DNase and RNase sensitivity. The strandedness and conformation (i.e., circular) of the genome can be determined by techniques known in the art, including visualization by electron microscopy and sedimentation characteristics.
Based on hybridization studies to cloned cDNA molecules derived from the N-(ABCDE) hepatitis agents by the method of the present invention, the nature of the genome can be further evaluated. If, for example, a N- (ABCDE) hepatitis genome is RNA, it can be determined from hybridization of the genomic RNA to the cDNA probes whether the genomic RNA is the positive or negative strand. The isolated genomes also make it possible to sequence the entire genome whether it is segmented or not, and whether it is an RNA or DNA genome (using, for example RT-PCR, chromosome walking techniques, or PCR which utilizes primers from adjacent cloned sequences) . Determination of the entire sequence of a N-(ABCDE) hepatitis agent allows genomic organization studies and the comparison of the N-(ABCDE) hepatitis sequences to the coding and regulatory sequences of known viral agents.
F. Screening for Agents Having Anti-N-(ABCDE) Hepatitis Activity.
The use of cell culture and animal model systems for propagation of N-(ABCDE) hepatitis agents provides the ability to screen for anti-hepatitis agents which inhibit the production of infectious N-(ABCDE) hepatitis agents: in particular, drugs that inhibit the replication of N- (ABCDE) hepatitis agents. Cell culture and animal models allow the evaluation of the effect of such anti-hepatitis drugs on normal cellular functions and viability.
Potential anti-viral agents (including, for example, small molecules, complex mixtures such as fungal extracts, and anti-sense oligonucleotides) are typically screened for anti-viral activity over a range of anti-viral agent concentrations. The effect on N-(ABCDE) hepatitis agent replication and/or antigen production is then evaluated relative to the effect of the anti-viral agent on normal cellular function (DNA replication, RNA transcription, general protein translation, etc.). The detection of the N-(ABCDE) hepatitis agent can be accomplished by the methods described in the present specification. For example, antibodies can be generated against the antigens of the present invention and these antibodies used in antibody-based assays (Harlow, et al . ) to identify and quantitate N-(ABCDE) hepatitis antigens in cell culture. N-(ABCDE) hepatitis antigens can be quantitated in culture using competition assays: polypeptides encoded by the cloned N-(ABCDE) hepatitis agent sequences can be used in such assays. Typically, a recombinantly produced N-(ABCDE) hepatitis antigenic polypeptide is produced and used to generate a monoclonal or polyclonal antibody. The recombinant N-(ABCDE) hepatitis polypeptide is labelled using a reporter molecule. The inhibition of binding of this labelled polypeptide to its cognate antibody is then evaluated in the presence of samples (e . g. , cell culture media or sera) that contain N-(ABCDE) hepatitis antigens. The level of N-(ABCDE) hepatitis antigens in the sample is determined by comparison of levels of inhibition to a standard curve generated using unlabelled recombinant proteins at known concentrations. The N-(ABCDE) hepatitis sequences of the present invention are particularly useful for the generation of polynucleotide probes/primers that may be used to quantitate the amount of N-(ABCDE) hepatitis nucleic acid sequences produced in a cell culture system. Such quantification can be accomplished in a number of ways. For example, probes labelled with reporter molecules can be used in standard dot-blot hybridizations or competition assays of labelled probes with infected cell nucleic acids. Further, there are a number of methods using the polymerase chain reaction to quantitate target nucleic acid levels in a sample (Osikowicz, et al . ) .
Neutralizing antibodies can also be identified using the cell culture and animal model systems described above. For example, polyclonal or monoclonal antibodies are generated against the antigens of the present invention. These antibodies are then used to pre-treat sera before infection of cell cultures or animals. The ability of a single antibody or mixtures of antibodies to protect the cell culture or animal from infection is evaluated. For example, in cell culture and animals the absence of viral antigen and/or nucleic acid production serves as a screen. Further in animals, the absence of N-(ABCDE) hepatitis disease symptoms, e.g., elevated ALT values, is also indicative of the presence of neutralizing antibodies.
Alternatively, convalescent sera can be screened for the presence of neutralizing antibodies and then these sera used to identify N-(ABCDE) hepatitis agent antigens that bind with the antibodies. The identified N-(ABCDE) hepatitis antigen is then recombinantly or synthetically produced. The ability of the antigen to generate neutralizing antibodies is tested as above. After initial screening, the antigen or antigens identified as capable of generating neutralizing antibodies, either singly or in combination, can be used as a vaccine to inoculate test animals. The animals are then challenged with infectious N-(ABCDE) hepatitis agents. Protection from infection indicates the ability of the animals to generate neutralizing antibodies that protect them from infection.
G. Vaccines and Neutralizing Antibodies. Vaccines can be prepared from one or more of the immunogenic polypeptides identified by the method of the present invention. Homologies between the isolated sequences from N-(ABCDE) hepatitis agents and other known viral proteins may provide information concerning the polypeptides that are likely to be candidates for effective vaccines. In addition, a number of computer programs can be used for to identify likely regions of isolated sequences that encode protein antigenic determinant regions (for example, Hopp, et al . ; "ANTIGEN," Intelligenetics, Mountain View CA) .
Vaccines containing immunogenic polypeptides as active ingredients are typically prepared as injectables either as solutions or suspensions. Further, the immunogenic polypeptides may be prepared in a solid or lyophilized state that is suitable for resuspension, prior to injection, in an aqueous form. The immunogenic polypeptides may also be emulsified or encapsulated in liposomes. The polypeptides are frequently mixed with pharmaceutically acceptable excipients that are compatible with the polypeptides. Such excipients include, but are not limited to, the following and combinations of the following: saline, water, sugars (such as dextrose and sorbitol) , glycerol, alcohols (such as ethanol [EtOH]), and others known in the art. Further, vaccine preparations may contain minor amounts of other auxiliary substances such as wetting agents, emulsifying agents (e . g. , detergents), and pH buffering agents. In addition, a number of adjuvants are available which may enhance the effectiveness of vaccine preparations. Examples of such adjuvants include, but are not limited to, the following: the group of related compounds including N-acetyl-muranyl- L-threonyl-D-isoglutamine and N-acetyl-nor-muranyl-L- alanyl-D-isoglutamine, and aluminum hydroxide.
The immunogenic polypeptides used in the vaccines of the present invention may be recombinant, synthetic or isolated from, for example, attenuated N-(ABCDE) hepatitis agent particles. The polypeptides are commonly formulated into vaccines in neutral or salt forms. Pharmaceutically acceptable organic and inorganic salts are well known in the art.
N-(ABCDE) hepatitis vaccines are parenterally administered, typically by subcutaneous or intramuscular injection. Other possible formulations include oral and suppository formulations. Oral formulations commonly employ excipients (e .g. , pharmaceutical grade sugars, saccharine, cellulose, and the like) and usually contain within 10-98% immunogenic polypeptide. Oral compositions take the form of pills, capsules, tablets, solutions, suspensions, powders, etc., and may be formulated to allow sustained or long-term release. Suppository formulations use traditional binders and carriers and typically contain between 0.1% and 10% of the immunogenic polypeptide. In view of the above information, multivalent vaccines against N-(ABCDE) hepatitis agents can be generated which are composed of one or more structural or non-structural viral-agent protein(s) . These vaccines can contain recombinantly prepared N-(ABCDE) hepatitis agent polypeptides and/or polypeptides isolated from N-(ABCDE) hepatitis agent virions. In addition, it may be possible to prepare vaccines, which confer protection against N- (ABCDE) hepatitis infection through the use of inactivated N-(ABCDE) hepatitis agents. Such inactivation might be achieved by preparation of viral lysates followed by treatment of the lysates with appropriate organic solvents, detergents or formalin.
Vaccines may also be prepared from attenuated N- (ABCDE) hepatitis agent strains. Such attenuated N- (ABCDE) hepatitis agents may be obtained utilizing the above described cell culture and/or animal model systems. Typically, attenuated strains are isolated after multiple passages in vitro or in vivo . Detection of attenuated strains is accomplished by methods known in the art. One method for detecting attenuated N-(ABCDE) hepatitis agents is the use of antibody probes against N-(ABCDE) hepatitis antigens, sequence-specific hybridization probes, or amplification with sequence-specific primers to screening in vivo or in vitro cultures.
Alternatively, or in addition to the above methods, attenuated N-(ABCDE) hepatitis strains may be constructed based on the genomic information that can be obtained from the information presented in the present specification. Typically, a region of the infectious agent genome that encodes, for example, a polypeptide that is related to viral pathogenesis can be deleted. The deletion should not interfere with viral replication. Further, the recombinant attenuated N-(ABCDE) hepatitis agent construct allows the expression of an epitope or epitopes that are capable of giving rise to neutralizing antibodies against the N-(ABCDE) hepatitis agent. The genome of the attenuated N-(ABCDE) hepatitis agent is then used to transform cells and the cells grown under conditions that allow viral replication. Such attenuated strains are useful not only as vaccines, but also as production sources of viral antigens and/or N-(ABCDE) hepatitis particles. Hybrid particle immunogens that contain N-(ABCDE) hepatitis epitopes can also be generated. The immunogenicity of N-(ABCDE) hepatitis epitopes may be enhanced by expressing the epitope in a eucaryotic systems (e . g. , mammalian or yeast systems) where the epitope is fused or assembled with known particle forming proteins. One such protein is the hepatitis B surface antigen. Recombinant constructs where the N-(ABCDE) hepatitis epitope is directly linked to coding sequence for the particle forming protein will produce hybrid proteins that are immunogenic with respect to the N-(ABCDE) hepatitis epitope and the particle forming protein. Alternatively, selected portions of the particle-forming protein coding sequence, which are not involved in particle formation, may be replaced with coding sequences corresponding to N- (ABCDE) hepatitis epitopes. For example, regions of specific immunoreactivity to the particle-forming protein can be replaced by N-(ABCDE) hepatitis epitope sequences. The hepatitis B surface antigen has been shown to be expressed and assembled into particles in the yeast Saccharomyces cerevisiea and in mammalian cells
(Valenzuela, et al., 1982 and 1984; Michelle, et al . ) . These particles have been shown to have enhanced immunoreactivity. Formation of these particles using hybrid proteins, i.e., recombinant constructs with heterologous viral sequences, has been previously disclosed (EPO 175,261, published 26 March 1986). Such hybrid particles containing N-(ABCDE) hepatitis epitopes may also be useful in vaccine applications.
The vaccines of the present invention are administered in dosages compatible with the method of formulation, and in such amounts that will be pharmacologically effective for prophylactic or therapeutic treatments. The quantity of immunogen administered depends on the subject being treated, the capacity of the treatment subject's immune system for antibody synthesis, and the desired level of protection. The amounts to be administered are usually determined by the administering health care professional.
The N-(ABCDE) hepatitis vaccines of the present invention can be administered in single or multiple doses. Dosage regimens are also determined relative to the treatment subject's needs and tolerances. In addition to the N-(ABCDE) hepatitis immunogenic polypeptides, vaccine formulations may be administered in conjunction with other immunoregulatory agents, such as immunoglobins.
H. Synthetic Peptides.
When the coding sequences of N-(ABCDE) hepatitis polypeptide antigens are determined synthetic peptides can be generated which correspond to these polypeptides. Synthetic peptides can be commercially synthesized or prepared using standard methods and apparatus in the art (Applied Biosystems, Foster City CA) .
Alternatively, oligonucleotide sequences encoding peptides can be either synthesized directly by standard methods of oligonucleotide synthesis, or, in the case of large coding sequences, synthesized by a series of cloning steps involving a tandem array of multiple oligonucleotide fragments corresponding to the coding sequence (Crea; Yoshio et al . ; Eaton et al.) . Oligonucleotide coding sequences can be expressed by standard recombinant procedures (Maniatis et al . ; Ausubel et al . ) .
V. Utility
A. Immunoassays for N-(ABCDE) Hepatitis Agents.
One utility for the antigens obtained by the methods of the present invention is their use as diagnostic agents for hepatitis antibodies present in N-(ABCDE) sera, thereby indicating current or past infections in the individual; in particular, D19, Clone 17A, Clone IA. The antigens of the present invention can be used singly, or in combination with each other, in order to detect single or multiple N-(ABCDE) hepatitis agents. In one diagnostic configuration, test serum is reacted with a solid phase reagent having a surface-bound antigen obtained by the methods of the present invention, e .g. , the D19 antigen. After binding anti-N-(ABCDE) hepatitis antibody to the reagent and removing unbound serum components by washing, the reagent is reacted with reporter-labelled anti-human antibody to bind reporter to the reagent in proportion to the amount of bound anti-N- (ABCDE) hepatitis antibody on the solid support. The reagent is again washed to remove unbound labelled antibody, and the amount of reporter associated with the reagent is determined. Typically, the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric or colorimetric substrate (Sigma, St. Louis, MO) . The solid surface reagent in the above assay is prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, 96-well plate or filter material. These attachment methods generally include non-specific adsorption of the protein to the support or covalent attachment of the protein, typically through a free amine group, to a chemically reactive group on the solid support, such as an activated carboxyl, hydroxyl, or aldehyde group. In a second diagnostic configuration, known as a homogeneous assay, antibody binding to a solid support produces some change in the reaction medium which can be directly detected in the medium. Known general types of homogeneous assays proposed heretofore include (a) spin- labelled reporters, where antibody binding to the antigen is detected by a change in reported mobility (broadening of the spin splitting peaks) , (b) fluorescent reporters. where binding is detected by a change in fluorescence efficiency, (c) enzyme reporters, where antibody binding effects enzyme/substrate interactions, and (d) liposome- bound reporters, where binding leads to liposome lysis and release of encapsulated reporter. The adaptation of these methods to the protein antigen of the present invention follows conventional methods for preparing homogeneous assay reagents.
In each of the assays described above, the assay method involves reacting the serum from a test individual with the protein antigen and examining the antigen for the presence of bound antibody. The examining may involve attaching a labelled anti-human antibody to the antibody being examined (for example from acute, chronic or convalescent phase) and measuring the amount of reporter bound to the solid support, as in the first method, or may involve observing the effect of antibody binding on a homogeneous assay reagent, as in the second method.
Also forming part of the invention is an assay system or kit for carrying out the assay method just described. The kit generally includes a support with surface-bound recombinant N-(ABCDE) hepatitis antigen (e.g., the D19 antigen, as above) , and a reporter-labelled anti-human antibody for detecting surface-bound anti-N-(ABCDE) antigen antibody.
A third diagnostic configuration involves use of the anti-N-(ABCDE) hepatitis antibodies capable of detecting N-(ABCDE) hepatitis specific antigens. The N-(ABCDE) hepatitis antigens may be detected, for example, using an antigen capture assay where N-(ABCDE) hepatitis antigens present in candidate serum samples are reacted with a N- (ABCDE) hepatitis specific monoclonal or polyclonal antibody. The antibody is bound to a solid substrate and the antigen is then detected by a second, different labelled anti-N-(ABCDE) hepatitis antibody. Antibodies can be prepared, utilizing the peptides of the present invention, by standard methods. Antibodies that are substantially'free of serum proteins which may affect reactivity can be generated (e . g. , affinity purification (Harlow et al . ) ) .
B. Hybridization Assays for N-(ABCDE) Hepatitis Agents.
One utility for the nucleic acid sequences obtained by the methods of the present invention is their use as diagnostic agents for hepatitis agent sequences present in N-(ABCDE) sera, thereby indicating infection in the individual. Primers and/or probes derived from the coding sequences of the present invention, in particular, D19, Clone 17A, Clone IA, and Clone 470-20-1 can be used singly, or in combination with each other, in order to detect single or multiple N-(ABCDE) hepatitis agents. In one diagnostic configuration, test serum is reacted under PCR or RT-PCR conditions using primers derived from, for example, 470-20-1 sequences. The presence of a N-(ABCDE) hepatitis agent, in the serum used in the amplification reaction, can be detected by specific amplification of the sequences targeted by the primers. Example 14 describes the use of polymerase chain amplification reactions, employing primers derived from the clones of the present invention, to screen different source material. The results of these amplification reactions demonstrate the ability of primers derived from the clones of the present invention (for example, 470-20- 1) , to detect homologous sequences by amplification reactions employing a variety of different source templates. The amplification reactions in Example 14 included use of nucleic acids obtained directly from sera samples as template material.
Alternatively, probes can be derived from the N- (ABCDE) sequences of the present invention. These probes can then be labelled and used as hybridization probes against nucleic acids obtained from test serum or tissue samples. The probes can be labelled using a variety of reporter molecules and detected accordingly: for example, radioactive isotopic labelling and chemiluminescent detection reporter systems (Tropix, Bedford, Mass.).
Also forming part of the invention is assay systems or kits for carrying out the amplification/hybridization assay methods just described. Such kits generally include either specific primers for use in amplification reactions or hybridization probes.
C. Therapeutic Uses. As discussed above, the N-(ABCDE) hepatitis antigens of the present invention can be used in vaccine preparation.
Further, antibodies generated against the polypeptide antigens of the present invention can be used for passive immunotherapy. The anti-N-(ABCDE) hepatitis antibodies of the invention can be used as a means of enhancing an anti- N-(ABCDE) hepatitis immune response since antibody-virus complexes are recognized by macrophages. The antibodies can be administered in amounts similar to those used for other therapeutic administrations of antibody. For example, pooled gamma globulin is administered at 0.02-0.1 ml/lb body weight during the early incubation of other viral diseases such as rabies, measles and hepatitis B to interfere with viral entry into cells. Thus, antibodies reactive with the N-(ABCDE) hepatitis antigens can be passively administered alone or in conjunction with another anti-viral agent to a host infected with a N- (ABCDE) hepatitis agent to enhance the immune response and/or the effectiveness of an antiviral drug.
The following examples illustrate, but in no way are intended to limit the present invention.
Materials and Methods E. coli DNA polymerase I (Klenow fragment) was obtained from Boehringer Mannheim Biochemicals (Indianapolis, IN) . T4 DNA ligase and T4 DNA polymerase were obtained from New England Biolabs (Beverly, MA) ; Nitrocellulose filters were obtained from Schleicher and Schuell (Keene, NH) .
Synthetic oligonucleotide linkers and primers were prepared using commercially available automated oligo¬ nucleotide synthesizers. Alternatively, custom designed synthetic oligonucleotides may be purchased from commercial suppliers. cDNA synthesis kit and random priming labeling kits were obtained from Boehringer-Mannheim Biochemical (BMB, Indianapolis, IN) or GIBCO/BRL (Gaithersburg, MD) .
Standard molecular biology and cloning techniques were performed essentially as previously described in Ausubel, et al . , Sambrook, et al . , and Maniatis, et al. Common manipulations involved in polyclonal and monoclonal antibody work, including antibody purification from sera, were performed by standard procedures (Harlow, et al . ) . Pierce or Promega (Madison, WI) were sources of many antibody reagents.
General ELISA Protocol for Detection of Antibodies.
Polystyrene 96 well plates Immulon II (PGC) were coated with 5 g/mL (100 μ per well) antigen in 0.1 M carb/bicarbonate buffer, pH 9.5. Plates are sealed with parafilm and stored at 4°C overnight.
Plates are aspirated and blocked with 300 uL 10% NGS and incubated at 37°C for 1 hr.
Plates were washed 5 times with PBS 0.5% "TWEEN- 20".
Antisera were diluted in 0.1 M PBS, pH 7.2. The desired dilution(s) of antisera (0.1 mL) were added to each well and the plate incubated 1 hours at 37°C. The plates was then washed 5 times with PBS 0.5% "TWEEN- 20".
Horseradish peroxidase (HRP) conjugated goat anti- human antiserum (Cappel) was diluted 1/5,000 in PBS. 0.1 mL of this solution was added to each well. The plate was incubated 30 min at 37°C, then washed 5 times with PBS.
Sigma ABTS (substrate) was prepared just prior to addition to the plate.
The reagent consists of 50 mL 0.05 M citric acid, pH 4.2, 0.078 mL 30% hydrogen peroxide solution and 15 mg ABTS. 0.1 mL of the substrate was added to each well, then incubated for 30 min at room temperature. The reaction was stopped with the addition of 0.050 mL 5% SDS (w/v) . The relative absorbance is determined at 410 nm.
EXAMPLE 1 CHARACTERIZATION OF N-fABCDE) SERA
The sera samples in the Table 1 were tested in standard ELISA assays using a number of known hepatitis antigens including the C100 Abbott test kit antigen (Abbott Laboratories, N. Chicago, IL) and HCV and HEV individual antigens. The 409-1-1 and the 33u antigens are from HCV NS3, antigen 36 from HCV NS5. All HCV epitopes, except antigen 36, were expressed as a fusion protein of glutathione-S-transferase (Smith, et al . ) . The original PNF 2161 inoculum was positive with HCV antigens. However, the My131 inoculated with PNF2161 tested negative for the presence of HCV by the RT-PCR assay, PCR using non-coding region HCV primers, and by ELISA with HCV antigens.
The presence of HCV nucleic acid was also tested using a reverse transcription polymerase chain reaction (Kawasaki, et al . ; Wang, et al . ) .
The sera PNF 2161, My 131, My 190, JFA, SCH, DEN and STA all test negative for the presence of antigens to HAV and HBV virus. Table 1
Figure imgf000045_0001
ND = not determined
* __= Hepatitis Source:
C: chronic hepatitis TX: transfusion A: acute hepatitis UK: unknown R: resolved
** ANTI-HEV negative in ELISA assays using 3-2 (M) , 4-2 (M) , 6-1-4 antigens, and 4-2 (B) (Yarbrough, et al . ) .
The HCV epitopes have been disclosed in Houghten, et al . , (C-100) and Reyes, et al . , (WO 91/15516,
"Hepatitis C Virus Epitopes", published 17 October
1991) .
EXAMPLE 2
CONSTRUCTION OF cDNA LIBRARIES One milliliter of each undiluted serum (JFA, PNF
2161, SCH, My 131, My 190, and DEN) was precipitated by the addition of PEG (MW 6,000) to 8% and centrifugation at 12K, for 15 minutes in an microfuge, at 4°C.
Alternatively, one milliliter of each undiluted serum (My 620 and My 670) was pelleted by centrifugation at 40,000 rpm in a type 70.1 rotor for 2 hours at 4°C. Each resulting pellet (by either of the above methods) was extracted for RNA and DNA. Half of the nucleic acids were converted to cDNA with random primer and reverse transcriptase after denaturation. The other half of each was converted to DNA with random primer and Klenow.
A. Isolation of RNA from Sera.
RNA was extracted from each resulting serum pellet essentially as described by Chomozynski. The pellet was treated with a solution containing 4M guanidine isothiocyanate, 0.18% 2- mercaptoethanol, and 0.5% sarcosyl. The treated pellet was extracted several times with acidic phenol-chloroform alcohol, and the RNA was precipitated with ethanol. This solution was held at -70°C for approximately 10 minutes and then spun in a microfuge at 4°C for 10 minutes. The resulting pellet was resuspended in 100 μl of DEPC- treated (diethyl pyrocarbonate) water, and 10 μl of NaOAc, pH=5.2, two volumes of 100% ethanol and one volume of 100% isopropanol were added to the solution. The solution was held at -70°C for at least 10 minutes. The RNA pellet was recovered by centrifugation in a microfuge at 12,000 x g for 15 minutes at 5°C. The pellet was washed in 70% ethanol and dried under vacuum.
B. Synthesis of cDNA
(i) First Strand Synthesis The synthesis of cDNA molecules was accomplished as follows. The above described RNA preparations were transcribed into cDNA, according to the method of Gubler et al . using random nucleotide hexamer primers (cDNA Synthesis Kit, BMB, Indianapolis, IN or GIBCO/BRL, Gaithersburg, MD) .
For nucleic acid samples derived from sera JFA, PNF 2161 and SCH, the nucleic acid pellet was treated with RNase-free DNase I (Ausubel, et al . ) prior to first strand synthesis; My 131 and My 190 sera were not treated with DNase.
After the second-strand cDNA synthesis, T4 DNA polymerase was added to the mixture to maximize the number of blunt-ends of cDNA molecules. The reaction mixture was incubated at room temperature for 10 minutes. The reaction mixture was extracted with phenol/chloroform and chloroform isoamyl alcohol. The cDNA was precipitated by the addition of two volumes of 100% ethanol and chilling at -70°C for 15 minutes. The cDNA was collected by centrifugation, the pellet washed with 70% ethanol and dried under vacuum.
C. Amplification of the Double Stranded cDNA Molecules.
The cDNA pellet was resuspended in 12 μl distilled water. To the resuspended cDNA molecules the following components were added: 5 μl phosphorylated linkers (Linker AB, a double strand linker comprised of SEQ ID NO:44 and SEQ ID NO:45, where SEQ ID NO:45 is in a 3' to 5' orientation relative to SEQ ID NO:44 — as a partially complementary sequence to SEQ ID NO:44), 2 μl lOx ligation buffer (0.66 M Tris.Cl pH=7.6, 50 mM MgCl2, 50 mM DTT, 10 mM ATP) and 1 μl T4 DNA ligase. Typically, the cDNA and linker were mixed at a 1:100 molar ratio in the presence of 0.3 to 0.6 Weiss units of T4 DNA ligase. The reaction was incubated at 14°C overnight. The following morning the reaction was incubated at 70°C for three minutes to inactivate the ligase.
To 100 μl of 10 mM Tris-Cl buffer, pH 8.3, containing 1.5 mM MgCl2 and 50 mM KC1 (Buffer A) was added about 1 x 10-3 μg of the linker-ligated cDNA, 2 μl of a primer having the sequence shown as SEQ ID
NO:44, 200 /i/M each of dATP, dCTP, dGTP, and dTTP, and 2.5 units of Thermus aquaticus DNA polymerase (Taq polymerase). The reaction mixture was heated to 94°C for 30 sec for denaturation, allowed to cool to 50°C for 30 sec for primer annealing, and then heated to 72°C for 0.5-3 minutes to allow for primer extension by Taq polymerase. The amplification reaction, involving successive heating, cooling, and polymerase reaction, was repeated an additional 25-40 times with the aid of a Perkin-Elmer Cetus DNA thermal cycler (Mullis; Mullis, et al . ; Reyes, et al . , 1991).
After the amplification reactions, the solution was then phenol/chloroform, chloroform/isoamyl alcohol extracted and precipitated with two volumes of ethanol. The resulting amplified cDNA pellets were resuspended in 20 μl TE (pH=7.5).
D. Cloning of the cDNA into Lambda Vectors. The linkers used in the construction of the cDNAs contained an EcoRI site which allowed for direct insertion of the amplified cDNAs into lambda gtll vectors (Promega, Madison WI or Stratagene, La Jolla, CA) . Lambda vectors were purchased from the manufacturer (Promega) which were already digested with EcoRI and treated with bacterial alkaline phosphatase, to remove the 5' phosphate and prevent self-ligation of the vector.
The .Eco.RJ-digested cDNA preparations were ligated into lambda gtll (Promega) . The conditions of the ligation reactions were as follows: 1 μl vector DNA (Promega, 0.5 mg/ml); 0.5 or 3 μl of insert cDNA; 0.5 μl 10 x ligation buffer (0.5 M Tris-HCl, pH=7.8; 0.1 M MgCl2; 0.2 M DTT; 10 mM ATP; 0.5 g/ml bovine serum albumin (BSA) ) , 0.5 l T4 DNA ligase (New England Biolabs) and distilled water to a final reaction volume of 5 j.1. The ligation reaction tubes were placed at 14°C overnight (12-18 hours) . The ligated cDNA was packaged the following morning by standard procedures using a lambda DNA packaging system (GIGAPAK, Stratagene, LaJolla, CA) , and then plated at various dilutions to determine the titer. A standard X-gal blue/white assay was used to determine recombinant frequency of the libraries (Miller; Maniatis et al . ) .
Percent recombination in each library was also determined as follows. A number of random clones were selected and corresponding phage DNA isolated. Polymerase chain reaction (Mullis; Mullis, et al . ) was then performed using isolated phage DNA as template and lambda DNA sequences, derived from lambda sequences flanking the EcoRI insert site for the cDNA molecules, as primers. The presence or absence of insert was evident from gel analysis of the polymerase chain reaction products.
E. cDNA Libraries Generated cDNA-insert phage libraries were generated from sera samples My 131, My 190, My 620, My 670, DEN, SCH, JFA and PNF 2161.
F. Deposit of cDNA Libraries.
The cDNA-insert phage libraries generated from sera samples My 131, My 190, DEN, SCH, JFA and PNF 2161 have been deposited with the American Type Culture Collection, 12301 Parklawn Dr., Rockville MD 20852, and have been assigned the following deposit designations: MY 131 cDNA source, ATCC 75273; PNF 2161 cDNA source, ATCC 75268; JFA cDNA source 75272; SCH cDNA source, ATCC 75283; DEN cDNA source, ATCC 75417; and MY 190, cDNA source, to be assigned. The cDNA- insert phage libraries generated from sera samples My 620 and My 670 can be obtained from Genelabs Technologies, Inc., 505 Penobscot Dr., Redwood City, CA 94063.
EXAMPLE 3 CONSTRUCTION OF DNA LIBRARIES
A. My 131 and My 190 Libraries, (i) Isolation of Nucleic Acids. Each sera, My 131 and My 190, was pelleted as described above. The resulting pellet was resuspended in 0.1 M NaCl, 50 mM Tris, pH 8, 1 mM EDTA, 0.5% SDS and treated with Proteinase K at a final concentration of 1 mg/ml. Nucleic acids were precipitated, after phenol/chloroform and chloroform/isoamyl alcohol extractions, by the addition of two volumes of ice-cold ethanol. This solution was held at -70°C for approximately 10 minutes and then spun in a microfuge at 4°C for 10 minutes.
The resulting pellet was resuspended in 100 μl of sterile TE (Maniatis, et al . ) . To this solution 10 μl of NaOAc, pH=5.2, and two volumes of 100% ethanol were added. The solution was held at -70°C for at least 10 minutes. The nucleic acid pellet was recovered by centrifugation in a microfuge at 12,000 x g for 15 minutes at 5°C. The pellet was washed in 70% ethanol, dried under vacuum, and resuspended in a minimum volume of TE.
(ii) Random Primer Synthesis From DNA Templates. The isolated nucleic acid was used as template for random primed DNA synthesis reactions. 2 μl of 10 x Klenow buffer (500 mM Tris-HCl, pH 7.5, 100 mM MgCl2, 10 mM DTT) , 2 μl of (0.5 mg/ml) hexanucleotide primer mixture (Boehringer Mannheim) . 2 μl of a 1.25 mM dNTP mixture and distilled water were added to the TE- resuspended nucleic acid to a final volume of 9 ul. The reaction mixture was heated to 95°C for 5 minutes to denature the DNA, and 1 μl of Klenow (1.5 u) was added to start the reaction upon cooling of the mixture. The reaction was placed at 37°C and typically carried out for 30 minutes. The reacted was stopped by the heat-inactivation of the Klenow enzyme at 65°C for 10 minutes. Blunt ends for the DNA molecules were generated by the treatment with the DNA polymerase as described previously.
Nucleic acids were precipitated and resuspended as described above.
(iϋ) Amplification and Cloning.
The random primed DNA mixture was ligated to linkers, PCR amplified and cloned in lambda gtll vectors as described in Example 2.
B. JFA, PNF 2161, SCH, DEN, My 620 and My 670 Libraries.
DNA libraries from sera JFA, PNF 2161, SCH, DEN, My 620 and My 670 were prepared essentially as described for the My 131 and My 190 libraries, except that, prior to random primer DNA synthesis, the nucleic acid samples were treated with DNase-free RNase (Boehringer Mannheim) (Ausubel, et al . ; Maniatis, et al . ) for JFA and PNF2161 libraries.
Further, for the preparation of DNA libraries from My 620 and My 670, initial pelleting of the sera was carried out as described in Example 2, i.e., pelleting by centrifugation instead of PEG precipitation.
C. Subtracted SCH DNA Libraries. (i) Isolation of Nucleic Acids.
One ml of SCH human serum was pelleted by ultracentrifugation at 210,000 g for 4 hours at 4°C. The resulting pellet was resuspended, treated, extracted and precipitated as described above. (ii) Random Primer Synthesis From DNA Templates.
The isolated nucleic acid was used as template for random primed DNA synthesis reactions, as described above. The samples were precipitated and resuspended as described above.
(iii) Amplification.
Normal human serum DNA was extracted from a healthy donor from Stanford blood bank, subjected to random priming and then ligated to C/D primer linker (nucleotide sequences of oligonucleotide C: SEQ ID NO:8 and of oligonucleotide D: SEQ ID NO:19). The resulting normal human DNA was 5' end-labelled with biotin using a biotinylated primer (at 1 μM) in a polymerase chain reaction. The reaction was performed as described in Example 2C except that the mixture was heated to 94°C for one minute, allowed to cool to 50°C for two minutes, and then heated to 72°C for 3 minutes and that the cycle was repeated 30 times.
In the presence of mineral oil, 0.1 μg of SISPA- amplified SCH DNA was incubated with 5 μg of the biotinylated normal human DNA in 50 μl of 0.12 M sodium phosphate buffer (pH 7.0) at 95°C for 5 minutes. The tube was then placed in a water bath heated to 55°C for 4 days. Streptavidin-conjugated paramagnetic particles ("DYNABEADS"; Dynal Inc., Lake Success, NY) suspended in 50 μl of 2x binding solution (2 M NaCl, 1 mM EDTA, 10 mM Tris-HCl, pH 7.5) were added to the solution containing the DNA, and the mixture was incubated at room temperature for 15 minutes.
The "DYNABEADS" were confined to one side of the reaction tube with a magnet, and the nearly particle- free solution ("supernatant") was decanted. The supernatant contained SISPA amplified DNA that had not hybridized with normal human DNA. (iv) Cloning.
The DNA in the supernatant from the above step was precipitated, resuspended in 20 μl TE, and cloned in lambda gtll vectors as described in Example 2. D. DNA Libraries Generated
DNA-insert phage libraries were generated from sera samples My 131, My 190, My 620, My 670, DEN, SCH, JFA and PNF 2161.
E. Deposit of DNA Libraries.
The DNA-insert phage libraries generated from sera samples My 131, My 190, DEN, SCH, JFA and PNF 2161 have been deposited with the American Type Culture Collection, 12301 Parklawn Dr., Rockville MD 20852, and have been assigned the following deposit designations: MY 131 DNA source, ATCC 75270; PNF 2161 DNA source, ATCC 75269; JFA DNA source, ATCC 75271; SCH DNA source, ATCC 75282; DEN DNA Source, ATCC 75418, and MY 190, DNA source, ATCC 75284. The DNA-insert phage libraries generated from sera samples DEN, My 620 and My 670, as well as subtracted SCH DNA libraries can be obtained from Genelabs Technologies, Inc. 505 Penobscot Dr., Redwood City, CA 94063.
EXAMPLE 4
IMMUNOSCREENING OF RECOMBINANT LIBRARIES
The lambda gtll libraries generated in Examples 1 and 2 were immunoscreened for the production of antigens recognizable by the five sera, or corresponding sera, from which the libraries were generated. The phage were plated for plaque formation using the Escherichia coli bacterial plating strain E. coli KM392 (Kevin Moore, DNAX, Palo Alto, CA) .
Alternatively, E. coli Y1090R- may be used.
The fusion proteins expressed by the lambda gtll clones were screened with serum antibodies essentially as described by Ausubel, et al . Each library was plated at approximately 2 x 104 phages per 150 mm plate. Plates were overlaid with nitrocellulose filters overnight. Filters were washed with TBS (10 mM, Tris pH 7.5; 150 mM NaCl) , blocked with AIB (TBS buffer, 10 mM Tris, pH 8.0, 150 mM NaCl; with 1% gelatin) and incubated with a primary antibody diluted 100 times in AIB. After washing with TBS, filters were incubated with a second antibody, goat-anti-human IgG conjugated to alkaline phosphatase (Promega, Madison, WI) . Reactive plaques were developed with a substrate (for example, BCIP, 5-bromo-4-chloro-3-indolyl-phosphate) , with NBT (nitro blue tetrazolium) salt (Sigma) .
Positive areas from the primary screening were replated and immunoscreened until pure plaques were obtained.
EXAMPLE 5 SEQUENCING OF THE cDNA INSERTS
OF IMMUNOREACTIVE CLONES
The cDNA inserts of immunoreactive lambda clones were subcloned into the "BLUESCRIPT SK+" vector
(Stratagene, LaJolla, CA) , pT7 Blue T vector (Novagen, Madison, WI) or TA cloning vector (Invitrogen, San
Diego, CA) . The sequences for the cDNA inserts were determined as per the manufacturer's instructions using the dideoxy chain termination technique (Sanger, 1979) .
Sequence data is presented in the Sequence
Listing. The sequences are typically presented with cloning linkers on each end. Sequences were compared with "GENBANK", EMBL database and dbEST (National Library of Medicine) sequences at both nucleic acid and amino acid levels. Search programs FASTA, BLASTP,
BLASTN and BLASTX indicated that these sequences are unique as both nucleic acid and amino acid sequences. In particular, none of the sequences presented in the Sequence Listing showed homology to any hepatitis virus for which sequence information is known. EXAMPLE 6
SOUTHERN BLOT ANALYSIS OF IMMUNOREACTIVE CLONES
The inserts of immunoreactive clones were screened for their ability to hybridize to the following control
DNA sources: normal human peripheral blood lymphocyte
DNA (purchased from Stanford Blood Bank) , normal mystax liver DNA (Centers for Disease Control) , and
Escherichia coli KM392 genomic DNA (Ausubel, et al . ; Maniatis, et al . ; Sambrook, et al . ) . Ten micrograms each of human lymphocyte DNA and normal mystax DNA, and 2 micrograms of E. coli DNA were digested with EcoRI and Hindlll. The restriction digestion products were electrophoretically fractionated on an agarose gel (Ausubel, et al . ) and transferred to nylon or nitrocellulose membranes (Schleicher and Schuell, Keene, NH) as per the manufacturer's instructions.
Probes from the immunoreactive clones were prepared as follows. Each clone was amplified using primers corresponding to lambda gtll sequences that flank the EcoRI cloning site of the gtll vector. Amplification was carried out by polymerase chain reactions utilizing each immunoreactive clones as template. The resulting amplification products were digested with EcoRI, the amplified fragments gel purified and eluted from the gel (Ausubel, et al . ) . The resulting amplified fragments, derived from the immunoreactive clones, were then random prime labelled (Boehringer Mannheim) using 32P-dNTPs. The random primed probes were then hybridized to the above-prepared nylon membrane to test for hybridization of the insert sequences to the control DNAs. Typically, inserts that hybridized with any of the control DNAs were removed from consideration. As positive hybridization controls, a probe derivative from a human C-kappa gene fragment (Hieter) was used as single gene copy control for human DNA and a E . coli polymerase gene fragment was similarly used for E. coli DNA.
EXAMPLE 7 SCREENING OF THE PNF 2161 LIBRARY A. Immunoscreening.
The cDNA and DNA libraries of PNF 2161 in lambda gtll were screened, as described in Example 4, with JFA, My 187 (Figure 1) and PNF 2161 sera. The results of the screening are presented in Table 2.
Table 2
PNF2161 LIBRARIES
Figure imgf000056_0001
1. Library constructed from the indicated human/mystax source, using either DNA or RNA as starting material.
2. Percent recombinant clones in the indicated λgtll library as determined by blue/white plaque assay and confirmed by PCR amplification of randomly selected clones.
3. Antisera source used for the immunoscreening of each indicated library. 4. Number of clones determined to have no immunoreactivity with pre-inoculu mystax and/or normal human sera.
One of the clones isolated by the above screen (PNF 2161 clone 470-20-1, SEQ ID NO:106), was used to generate extension clones, as described in Example 7C.
B. Sucrose Density Gradient Separation of PNF2161.
A continuous gradient of 10-60% sucrose ("ULTRAPURE", Gibco/BRL, Gaithersburg, MD) in TNE (50 mM Tris-Cl, pH 7.5, 100 mM NaCl, 1 mM EDTA) was prepared using a gradient maker from Hoefer Scientific (San Francisco, CA) . Approximately 12.5 ml of the gradient was overlaid with 0.4 ml of PNF serum which had been stored at -70°C, rapidly thawed at 37°C, then diluted in TNE. The gradient was then centrifuged in the SW40 rotor (Beckman) at 40,000 rpm (approximately. 200,000 x g at rlv) at 4°C for approximately 18 hours. Fractions of volume approximately 0.6 ml were collected from the bottom of the tube, and 0.5 ml was weighed directly into the ultracentrifuge tube, for calculation of density.
Table 3
MEASURED DENSITIES OF PNF FRACTIONS AND PRESENCE OF 470-20-1
Fraction Density 470-20-1 Detected*
1 1.274 —
2 1.274 -
3 1.266 -
4 1.266 -
5 1.260 -
6 1.254 - Fraction Density 470-20-1 Detected*
7 1.248 +
8 1.206 +
9 1.146 +
10 1.126 +++
11 1.098 ++++
12 1.068 +++
13 1.050 +
14 1.034 +
15 1.036 +
16 1.018 -
17 1.008 +
18 1.020 +
* "+" and "-" scores were initially based on 40-cycle PCR. In order to distinguish "+", "++", "+++", and "++++", fractions giving initial positive scores (7-18) were amplified with 30 cycles of PCR.
The putative viral particles were then pelleted by centrifugation at 40,000 rpm in the Ti70.l rotor (approximately 110,000 x g) at 4°C for 2 hours, and RNA was extracted using the acid guanidinium phenol technique ("TRI REAGENT", Molecular Research Center, Cincinnati, OH) , and alcohol-precipitated using glycogen as a carrier to improve recovery. The purified nucleic acid was dissolved in an RNase-free buffer containing 2 mM DTT and 1 U/ml recombinant RNasin. Analysis of the gradient fractions by RNA PCR showed a distinct peak in the 470-20-1 specific signal, localized in fractions of density ranging from 1.126 to 1.068 g/ml (Table 3). The 470-20-1 signal is thus shown, under these conditions, to form a discrete band, consistent with the expected behavior of a viral particle in a sucrose gradient.
C. Generation of 470-20-1 Extension Clones. The extracted RNA was passed through a "CHROMA SPIN" 100 gel filtration column (Clontech, Palo Alto, CA) to remove small molecular weight impurities. cDNA was synthesized using a Boehringer-Mannheim cDNA synthesis kit (Boehringer-Mannheim, Indianapolis, IN) . After cDNA synthesis the PNF cDNA was ligated to a 50 to 100 fold excess of KL-l/KL-2 SISPA linkers (SEQ ID NO:64, SEQ ID NO:65, respectively) and amplified for 35 cycles using the primer KL-1.
The 470 extension clones were generated by anchored PCR of a 1 μl aliquot from a 10 μl ligation reaction containing EcoRI digested (dephosphorylated) lambda gtll arms (1 μg) and EcoRI digested PNF cDNA (0.2 μg) . PCR amplification (40 cycles) of the ligation reaction was carried out using the lambda gtll reverse primer in combination with either 470-20-77F (SEQ ID NO:25) or 470-20-1-211R (SEQ ID NO:26). All primer concentrations for PCR were 0.2 μM. The amplification products (9 μl/100 μl) were separated on a 1.5% agarose gel, blotted to "NYTRAN" (Schleicher and Schuell, Keene, NH) , and probed with a digoxygenin labelled oligonucleotide probe specific for 470-20-1. The digoxygenin labeling was performed according to the manufacturer's recommendations using terminal transferase (Boehringer-Mannheim) . Bands that hybridized were gel-purified, cloned into the "TA
CLONING VECTOR pCR II" (Invitrogen, San Diego, CA) , and sequenced. Clones having both 5' and 3' extensions were identified (designated 470F-16 and 470R-1, respectively) . All sequences reported here were confirmed by the sequencing of at least two independent isolates. The 5' extension obtained was 159 nucleotides and the 3' extension was 68 nucleotides (SEQ ID NO:104) .
EXAMPLE 8 IMMUNOSCREENING OF THE Mv 131 AND Mv 190 LIBRARIES A. Immunoscreening
The cDNA and DNA libraries of My 131 in lambda gtll were screened, as described in Example 4, with My 136 (Figure 1) and PNF 2161 sera. The results of the screening are presented in Table 4.
Table 4
Myl31 Libraries
% # # Clones #
Library1 Recom .2 Antibody3 Screened Plaque- Pre/Norm Purified & SB Neg4
Myl31/RNA 85 PNF 1.6X105 17 1
Myl31/DNA 91 PNF 1.2X105 34 7
Myl31/RNA 93 Myl36 6.0X104 11 1
Myl31/DNA 85 My136 6.0X104 44 8
TOTALS: 106 17
Library constructed from the indicated mystax source, using either DNA or RNA as starting material.
Percent recombinant clones in the indicated λgtll library as determined by blue/white plaque assay and confirmed by PCR amplification of randomly selected clones.
Antisera source used for the immunoscreening of each indicated library.
4. Number of clones determined to have no immunoreactivity with pre-inoculu mystax and/or normal human sera as well as an absence of hybridization to human mystax, and E. coli genomic DNA in Southern blotting (S.B.) assays.
The cDNA and DNA libraries of My 190 in lambda gtll were screened, as described in Example 4, with My 88 sera. The results of the screening are presented in Table 5.
Table 5
MyGB Libraries
% # # Clones #
Library1 Recomb.2 Antibody3 Screened Plaque- Pre/Norm Purified & SB
Neg4
MyGB/RNA 85 My88 1.5X105 19 6
MyGB/DNA 84 My88 1.1X10S 78 40
TOTALS: 97 46
Library constructed from the indicated human/mystax source, using either DNA or RNA as starting material.
Percent recombinant clones in the indicated λgtll library as determined by blue/white plaque assay and confirmed by PCR amplification of randomly selected clones.
Antisera source used for the immunoscreening of each indicated library.
Number of clones determined to have no immunoreactivity with pre-inoculum mystax and/or normal human sera as well as an absence of hybridization to human, mystax, and E. coli genomic DNA in Southern blotting (S.B.) assays. B. Characterization of Mv 131 Clones All clones that (i) tested positive for immunoreactivity in the plaque screening assays using PNF 2161 and My 136 sera, (ii) were determined to be exogenous to human, mystax, and E. coli genomic DNA, and (iii) were non-immunoreactive with pre-inoculum and/or normal human sera, were subcloned, sequenced and further analyzed.
Seven candidate clones were found to have open reading frames in phase with the β-galactosidase - 427- 7-1, 428-2-3, 428-7-3, 428-3-1, 430-4-8, 430-2-1 and 430-3-4.
C. Characterization of Mv 190 Immunoreactive Clones
All clones that (i) tested positive for immunoreactivity in the plaque screening assay using My 88 sera, (ii) were determined to be exogenous to human, mystax, and E. coli genomic DNA, and (iii) were non- immunoreactive with pre-inoculum and/or normal sera, were subcloned, sequenced and further analyzed.
Seven primary candidate clones were found having open reading frames continuous with the 3-galactosidase - D5, D6, D12, D13, D19, D44, and D76. Eleven additional clones contained open reading frames greater than 15 amino acids in length. The are: D20, D24, R27, D30, D31-2, D48, and D64.
Two of the My 190 immunoreactive clones were rescreened by the plaque screening assay against the sera shown in Table 6. Table 6
Sera
D19 D30
My73 pre-GB - —
My73 post-GB IgM + -
IgG +
My136 pre-GB - -
Myl36 post-GB - -
My242 pre-GB - -
My242 post-GB - -
My88 + +
One of the clones, D19, showed cross-reactivity with other mystax hepatitis N(ABCDE) sera.
The sera used in Table 6 were characterized as follows (refer to Figures 1 and 2): MY 73 and MY 136, were initially infected with 1 ml of 10% liver homogenate of MY 131 treated with CHC13. 100 days after infection, MY 73 and MY 136 were cross challenged with GB agent; MY 242, infected with 10% liver homogenate of MY 131 (not treated with CHC13) , was cross challenged with GB agent after 100 days; MY 190 and MY 88 are, respectively, the acute phase and convalescent phase sera for GB agent; MY 162 is a normal mystax serum; MY 131 is the acute phase mystax serum for PNF2161; and PNF2161 is a chronic human hepatitis serum. "Pre-GB" means 100 days after inoculation with MY 131 liver homogenate, that is, convalescent phase sera that corresponds to the MY 131 inoculum. EXAMPLE 9
FURTHER CHARACTERIZATION OF CLONES D6 and D19
A. Amplification Reactions with Genomic DNA using D19 Primers
Primers were derived from the insert of clone D19
(D19-NF, SEQ ID NO:68; D19-BR, SEQ ID NO:69). These primers were used in polymerase chain reaction amplifications (Mullis; Mullis, et al . ) with the following substrate DNAs: normal human (peripheral lymphocyte DNA, Stanford Blood Bank) , normal mystax liver DNA (Mystax 753, Centers for Disease Control), and E . coli (KM392) . Two controls were also run, (i) e no substrate DNA control and (ii) a positive control corresponding to the plasmid containing the cloned DNA from which the primer set was derived (i.e., plasmid D19) . The results of the amplification reactions are presented in Table 7.
Table 7
Figure imgf000064_0001
The results presented in Table 7 further demonstrate that cloned sequence D19 is not derived from normal human, mystax, or bacterial genomes, i.e., D19 represents sequences exogenous to these test genomic DNAs. B. Further Immunoscreening using D19.
The D19 clone encoded antigen was immunoscreened against nine additional mystax serum samples. The clone was immunoreactive with 2/9 of the sera.
C. Expression and Partial Purification of D6 and D19 as GST Fusion Proteins
1. Expression of D6 and D19 Fusion Proteins. The D6 and D19 sequences were subcloned into the bacterial expression vector, pGEX-GLI. The pGEX-GLI is a modification of the pGEX-1 vector of Smith, et al . , which involved the insertion of a thrombin cleavage sequence in-frame with the glutathione-S-transferase protein (GST: sj26 coding sequence) and addition of
NcoJ and BamHI restriction sites. The WcoJ primers in the amplified fragment allow in-frame fusion of D6 or D19 coding sequence to the sj26-thrombin coding sequences. The D19 coding sequence insert was generated by the polymerase chain reaction using PCR primers specific for each insert. Typically, the 5' primer contains a Ncol restriction site and the 3' primer contains stop-codons followed by a BamHI restriction site. The D19-5' primer was D19-NF (SEQ ID NO:68). The D19-3' primer was D19-RF (SEQ ID NO:69). The generated PCR product was digested with Ncol and BamHI and gel purified.
D6 coding sequence insert was prepared in a similar fashion. The D6-5' primer was D6-NF (SEQ ID NO:66) and the D6-3' primer was D6-BR (SEQ ID NO:67). The pGEX-GLI vector was digested with Ncol and BamHI and the linear vector isolated.
Manipulations were carried out for both coding sequence inserts essentially as described below for the D19 insert sequence. The Ncol /BamHI D19 fragment was ligated to the linear pGEX-GLI vector. The ligation mixture was transformed into E . coli and ampicillin resistant colonies were selected. Plasmids were isolated from the ampicillin resistant colonies and analyzed by restriction enzyme digestion. One of the candidate clones was designated pGEX-GLI-D19.
E. coli strain JM101 was transformed with pGEX- GLI-D19 and was grown at 37°C overnight. DNA was prepared from randomly-picked colonies. The presence of the insert coding sequence was confirmed by (i) restriction digest mapping and (ii) hybridization screening using labelled D19 inserts (i.e.. Southern analysis) .
2. Partial Purification of D6 and D19. Partial purification of the D19 encoded protein is described below: D19 protein was purified using the same methodology.
A D19 clone was identified, see above, and grown overnight. The overnight culture was diluted 1:10 with LB medium containing ampicillin and grown for one hour at 37°C. Alternatively, the overnight culture was diluted 1:100 and grown to CD of 0.5-1.0 before addition of IPTG (isopropylthio-j8-galactoside) . IPTG
(GIBCO-BRL) was added to a final concentration of 0.2- 0.5 mM for the induction of protein expression and the incubation was continued for 2-5 hours, preferably 3.5 hours.
Bacterial cells were harvested by centrifugation and resuspended in 1/100 culture volume of MTPBS (150 mM NaCl, 16 mM Na2HP04, 4 mM NaH2P04) . Cells were lysed by lysozyme, sonication or French press, and lysates cleared of cellular debris by centrifugation.
Sometimes, an aliquot of the supernatant obtained from IPTG-induced cultures of pGEX-GLI-Dl9-containing cells and an aliquot of the supernatant obtained from
IPTG-induced cultures of pGEX-GLI-vector alone were analyzed by SDS-polyacrylamide gel electrophoresis followed by Western blotting, as described below.
If necessary, the extracts can be concentrated by ultrafiltration using, for example, a "CENTRICON 10" filter.
Alternatively, the fusion proteins were partially purified over a glutathione agarose affinity column as described in detail by Smith, et al . In this method, 100 ml cultures were grown overnight. The cultures are diluted to 1 liter, and the cells grown another hour at 37°C. Expression of the fusion proteins was induced using IPTG. The induced cultures were grown at 37°C for 3.5 hours. Cells were harvested and a sonicator was used to lyse the cells. Cellular debris was pelleted and the clear lysate was loaded onto a glutathione "SEPHAROSE" column. The column was washed with several column volumes. The fusion protein was eluted from the affinity column with reduced glutathione and dialyzed.
D. Western Blot Analysis
Six milliliters of the above culture media were concentrated to 50 μl using a "CENTRICON-10" micro- concentrator (Amicon) . The concentrated media was brought to a total volume of 400 μl by addition of
SDS-ME disruption buffer and 10 μl was loaded into each of four lanes of a 12% reducing acrylamide gel (Ausubel et al . ) . Protein was transferred using standard procedures (Ausubel et al . ) from the gel to nitrocellulose membrane (Schleicher & Schuell) .
The membranes were incubated with the test serum (for example, Mystax 88 or Ruf serum) and washed. The membranes were then incubated with a labelled antibody suitable for detection of binding of antibodies from the test sera: for example, alkaline phosphatase- conjugated goat anti-human antibody (Mystax 88) . Excess goat anti-human IgG antibody was removed from the membranes (Ausubel et al . ; Harlow et al . ) and the membranes colori etrically developed.
Figure 3 illustrates the results of a Western blot analysis of the membrane containing crude lysates of the following samples: induced (Figure 3, lane 1) and un-induced (Figure 3, lane 2) pGEX-GLI-D6; induced (Figure 3, lane 3) and un-induced (Figure 3, lane 4) pGEX-GLI-D19. For the blot, the test serum was a 1:100 dilution of N-(ABCDE) mystax (My 88) serum. Figures 4A and 4B illustrates Western blots of the partially purified proteins using mystax (My 88) serum (Figure 4A) and N-(ABCDE) human (Ruf) serum (Figure 4B) . The partially purified proteins include D6 and D19 fusion proteins. The sj26 antigen was used as a control.
As shown in the Figures 3 and 4, the membranes treated with infected sera (My88 and human (Ruf) ) showed specific immunoreactivity with the N-(ABCDE) fusion proteins and Ruf serum reacted weakly to Sj26. The pGEX-GLI-D19 is considered negative with the human serum.
EXAMPLE 10 IMMUNOSCREENING OF THE JFA LIBRARY A. Immunoscreening
The cDNA and DNA libraries of JFA in lambda gtll were constructed and screened, as described in Example 2, 3 and 4, with JFA sera. The results of the screening are presented in Table 8. Table S
# Clones #
% Anti¬ # Plaque - Norm & SB
Library1 Recomb.2 body3 Screened Purified Neg.4
JFA/RNA (<500 bp) 95 JFA 1.5X105 0 0
JFA/DNA (<500 bp) 95 JFA 1.0X105 4 2
JFA/DNA (>500 bp) 91 JFA 1.0X105 7 5
TOTALS: 11 7
1. Library constructed from the indicated human source, using either DNA or RNA as starting material.
Percent recombinant clones in the indicated λgtll library as determined by blue/white plaque assay and confirmed by PCR amplification of randomly selected clones.
3. Antisera source used for the immunoscreening of each indicated library.
4. Number of clones determined to have no immunoreactivity with normal human sera as well as an absence of hybridization to human genomic DNA in Southern blotting (S.B.) assays.
B. Characterization of JFA Clones
All clones that (i) tested positive for immunoreactivity in the plaque screening assays using JFA, (ii) were determined to be exogenous to human DNA, and (iii) were non-immunoreactive with normal sera, were subcloned, sequenced and further analyzed. In addition, one clone containing sequences that hybridized with human sequences but that also had broad cross reactivity with N-(ABCDE) infected sera was sequenced (clone 8A) .
Three candidate clones (IA, 4B(a,b,c), and 17A) were found having large open reading frame continuous with the /3-galactosidase. 4B(a,b,c) is a co-ligated DNA clone consisting of three inserts separated by cloning linkers.
The characterization of the JFA clones is summarized in Table 9.
Table 9
Figure imgf000070_0001
Y = 1) Presence of ORF
2) Absence of known homology to known sequence
3) Exogenous clone
N = 1) Absence of continuous ORF
2) Homology to known sequences in data base
3) Human genomic sequence
N.D. = Not determined
*1A, 17A expressed in pGEX-GLI in E. coli . Insoluble proteins.
C. Further Characterization of Clones 17A and IA Clones 17A and IA have both been shown to have exogenous, non-human insert sequences. Both clones contain a single coding sequence continuous with the open reading frame of -galactosidase in the lambda gtll vector.
The insert-coding sequences of both clones 17A and IA have been cloned into the pGEX-GLI vector and expressed as fusion proteins, as described above for clones D6 and D19. For the expression of JFA 17A the following primers were used: 17A-NF, 5' (SEQ ID NO:70) and 17A-BR, 3' (SEQ ID NO:71). For the expression of JFA IA the following primers were used: 1A-NF, 5' (SEQ ID NO:72) and 1A-BR, 3' (SEQ ID NO:73) .
The following samples were electrophoretically separated on a polyacrylamide gel: Sj26 (lanes 2, 3, 10 and 11) ; the induced crude lysates from clones 17A and IA — lanes 4-8 and 12-15, respectively, where (i) lanes 4 and 12 are from zero time points, (ii) lanes 5- 8 are from a 3.5 hour induced sample, with increasing protein concentration per lane, and (iii) lanes 13-15 are from a 2 hour induced sample, with increasing protein concentration per lane; and a pool of purified clone IA protein before (lane 16) and after (lane 17) concentration using a stirred-gel micro-concentrator (Amicon) . Sj26 as a control. Lanes 1 and 9 contained molecular weight standards. The gel was coomassie blue stained. A copy of the photograph of the stained gel is presented as Figure 5A.
Figure 5B shows a Western blot analysis of the Figure 5A polyacrylamide gel transferred to a membrane and probed with JFA serum. As can be seen in Figure 5B, both the SJ26-17A and SJ26-1A fusion proteins are immunoreactive with JFA serum; however, equivalent amounts of Sj26 protein are not immunoreactive. In Figure 5B the arrows indicate the locations of the SJ26-17A and SJ26-1A fusion proteins, first and second panels, respectively. Figure 5C shows the Western blot analysis of Figure 5A polyacrylamide gel transferred to a membrane and probed with normal human serum. As can be seen in Figure 5C, IA and 17A proteins are not immunoreactive to normal serum. D. Isolation of Overlapping Clones to Clones 17A and IA
A JFA DNA library was generated in "LAMBDA ZAPII" (Stratagene, LaJolla, CA) using JFA random-primed SISPA DNA, prepared essentially as described in Example 3, except that the amplified DNA was digested with WotJ instead of EcoRI.
Undigested "LAMBDA ZAPII" DNA was cut with NotI and then treated with calf intestinal phosphatase (CIP) to prevent re-ligation of phage DNA ends.
A ligation reaction to insert JFA SISPA DNA fragments into phage DNA at the NotJ site was performed essentially as described in Example 2D. The ligated cDNA was packaged the following morning by standard procedures using a lambda DNA packaging system
(Stratagene) . The packaged phage were used to infect E . coli strain PLK-A' . A titer for the recombinant DNA phage library was determined by standard methods.
E. Screening of Overlapping Clones to Clone 17A About 4 x 105 "LAMBDA ZAP II" recombinants were screened using radioactively-labelled insert of clone 17A. Specifically, EcoRI DNA inserts of clone 17A were labelled by random-priming (Boehringer Mannheim) and employed as a probe in hybridization experiments to identify overlapping DNA sequences. Sequences identified by this method can, in turn, be used as probes to identify further contiguous clones.
In a primary screening of the random-primed JFA library using the clone 17A insert (screening was performed essentially as described in Example 6) , 8 hybridization-positive clones were identified.
The hybridization-positive clones were screened with oligonucleotides located at 5'- or 3'-ends of clone 17A. To screen clone 17A positives the 5'-end oligonucleotide was 17A-57F, 5' (SEQ ID NO:56) and the 3'-end oligonucleotide was 17A-602R, 3' (SEQ ID N0:57). The oligonucleotides were end-labelled with T4 polynucleotide kinase and 7~32P-ATP (Maniatis, et al . )
The screening procedure using oligonucleotides complementary to clone 17A resulted in l positive and 7 negatives. The clone that showed a positive hybridization to the 3'-end primer contained an insert of about 400 base pairs as estimated by gel electrophoresis. This clone was designated clone WT54. The insert of WT54 was sequenced and contained a 210 base pair overlap with 17A which extends 119 base pairs from the 3'-end of 17A. The sequence of this insert is presented as SEQ ID NO:58. Figure 6 schematically illustrates the overlap between clone 17A and WT54.
The 17A clones that gave a negative hybridization result with the oligonucleotide primers were rescreened. The probe used for rescreening was a PCR- generated clone 17A insert generated from 5'-and 3'-end oligonucleotide primers (SEQ ID NO:70 and SEQ ID NO:71) , and digested with Ncol and BamHI prior to labelling. This procedure resulted in 6 positives and 1 negative. All 6 positive clone inserts were sequenced and corresponded to internal 17A sequences.
F. Characterization of WT54 Clone The WT54 clone was characterized for its ability to hybridize to the following control DNA sources: normal human peripheral blood lymphocyte DNA (purchased from Stanford Blood Bank) , normal mystax liver DNA (Centers for Disease Control) , and Escherichia coli KM392 genomic DNA (Ausubel, et al . ; Maniatis, et al.; Sambrook, et al.) . Ten micrograms each of human lymphocyte DNA and normal mystax DNA, and 2 micrograms of E. coli DNA were digested with EcoRI and Hindlll. The restriction digestion products were electrophoretically fractionated on an agarose gel
(Ausubel, et al . ) and transferred to nylon membranes (Schleicher and Schuell, Keene, NH) as per the manufacturer's instructions.
Radiolabelled clone WT54 was prepared as follows. Oligonucleotide 5'- and 3'- primers were synthesized in the region of clone WT54 that does not overlap with clone 17A. The 5'-primer is WT54-590F (SEQ ID NO:59) and the 3'-primer is WT54-684R (SEQ ID NO:60). The primers were used to amplify the region between base pairs 590 and 684 by PCR. The resulting amplified fragment was then labelled by the random-priming method (Boehringer Mannheim) using 32P-dNTPs and then used as a probe.
The labelled probe was then hybridized to the above-prepared nylon membrane to test for hybridization of the insert sequences to the control DNAs. The probe did not hybridize to human, E. coli or mystax genomic DNA.
To determine that the 17A-WT54 linked sequence is present in JFA SISPA DNA and is not an artifact due to cloning manipulations, the following primers were selected, prepared and used in amplification reactions: a 5'-primer unique to clone 17A sequences, i.e., 17A sequences that do not overlap WT54 sequences; and a 3'- primer unique to clone WT54 sequences, i . e . , WT54 sequences that do not overlap 17A sequences. The 5'- primer is 17A-215F (Figure 7, A — SEQ ID NO:61) and the 3'-primer is WT54-684R (Figure 7, B — SEQ ID NO:60). The primers were used to amplify the putative linked region between base pairs 215 and 684 by PCR using JFA SISPA DNA and serum DNA as template.
The resulting amplified product was then amplified using internal nested primers, 17A-258F (Figure 7, C — SEQ ID NO:62) and WT54 647R (Figure 7, D — SEQ ID NO:63). The final PCR product was probed with a labelled-oligonucleotide specific for a non-overlapping 17A insert sequence, 17A-312F (Figure 7, Probe 1 — SEQ ID NO:52), and a labelled-oligonucleotide specific to a non-overlapping WT54 sequence, WT54-592F (Figure 7, Probe 2 — SEQ ID NO:53).
Hybridization screening of the nested amplification products, using JFA SISPA DNA as template, with Probes 1 and 2 gave a positive result. This result indicates that JFA 17A and WT54 sequences are contiguous in the JFA SISPA DNA.
EXAMPLE 11 IMMUNOSCREENING OF THE SCH LIBRARIES
A. Immunoscreenin .
The unsubtracted and subtracted SCH DNA libraries, generated from SCH serum in lambda gtll, were screened with convalescent SCH serum as described in Example 4. The DNA clones obtained from the unsubtracted library were designated "SU". The DNA clones from the subtracted DNA library of SCH were designated "SC". The results of the screening are presented in Table 10.
Table 10
Antibody2 # # Clones Exogenous
Library % Screened Plaque- Clones3
Recomb1 Purified
SU 83 SCH 1.6X105 32 17 (conval)
SC 94 SCH 2X105 27 2 (conval)
1. Percent recombinant clones in the indicated λgtll library as determined by blue/white plaque assay and confirmed by PCR amplification of randomly selected clones.
2. Antisera source used for the immunoscreening of each indicated library. 3. Number of clones determined to have no immunoreactivity with normal human sera as well as an absence of hybridization to human mystax, and E . coli genomic DNA in Southern blotting (S.B.) and genomic PCR assays.
B. Characterization of SCH Clones Nineteen clones (i) tested positive for immunoreactivity in the plaque screening assays using SCH, (ii) were determined to be exogenous to human, E. coli and yeast genomic DNA by genomic PCR (Example 14) , and (iii) were non-immunoreactive with normal sera. Further, the insert sequences were determined to be novel (i.e., they did not have sequence homology to any GENBANK sequences) and each contained an open reading frame continuous with the 3-galactosidase coding sequence of the lambda gtll vector.
These clones are identified by SEQ ID NO:84 to SEQ ID NO:102.
EXAMPLE 12 Isolation of D19 Fusion Protein Sepharose 4B beads conjugated with anti-?- galactosidase is purchased from Promega. The beads are packed in 2 ml column and washed successively with phosphate-buffered saline with 0.02% sodium azide and 10 ml TX buffer (10 mM Tris buffer, pH 7.4, 1% aprotinin) .
Lysogens infected with gtll/D19 are used to inoculate 500 ml of NZYDT broth. The culture is incubated at 32°C with aeration to an O.D. of about .2 to .4, then brought to 43°C quickly in a 43°C water bath for 15 minutes to induce gtll peptide synthesis, and incubated further at 37°C for 1 hour. The cells are pelleted by centrifugation, suspended in 10 ml of lysis buffer (10 mM Tris, pH 7.4 containing 2% "TRITON X-100" and 1% aprotinin added just before use. The resuspended cells are frozen in liquid nitrogen, then thawed, resulting in substantially complete cell lysis. The lysate is treated with DNase I to digest bacterial and phage DNA, as evidenced by a gradual loss of viscosity in the lysate. Non-solubi- lized material is removed by centrifugation.
The clarified lysate material is loaded on the Sepharose column, the ends of the column closed, and the column placed on a rotary shaker for 2 hrs. at room temperature and 16 hours at 4°C. After the column settles, it is washed with 10 ml of TX buffer. The fused protein is eluted with 0.1 M carbonate/bicarbonate buffer, pH 10. Typically, 14 ml of the elution buffer is passed through the column, and the fusion protein is eluted in the first 4-6 ml of eluate.
The eluate containing the fusion protein is concentrated in "CENTRICON-30" cartridges (Amicon, Danvers, Mass.). The final protein concentrate is resuspended in, for example, 400 μl PBS buffer. Protein purity is analyzed by SDS-PAGE.
EXAMPLE 13 Preparation of Anti-D19 Antibody Expression of a glutathione S-transferase fused protein (Sj26 fused protein) containing the D19 peptide antigen was achieved in E. coli strain JM101 (above) . The fusion protein is isolated from lysed bacteria, and isolated by affinity chromatography on a column packed with glutathione-conjugated beads, according to published methods (Smith, et al . ) .
The purified SJ26/D19 fused protein is injected subcutaneously in Freund's adjuvant in a rabbit. Approximately 1 mg of fused protein is injected at days 0 and 21, and rabbit serum is typically collected at 6 and 8 weeks. A second rabbit is similarly immunized with purified Sj26 protein obtained from control bacterial lysate.
Minilysates from the following bacterial cultures are prepared: (1) KM392 cells infected with pGEX and pGEX containing the D19 insert; and (2) cells infected with lambda gtll containing the D19 insert. The minilysates and a commercial source /3-galactosidase are fractionated by SDS-PAGE, and the bands transferred to nitrocellulose filters for Western blotting. Summarizing the expected results, serum from control (Sj26) rabbits is immunoreactive with each of the Sj26 and Sj26 fused protein antigens. Serum from the animal immunized with SJ26/D19 fused protein is reactive with all Sj-26 and ?-gal fusion proteins containing D19 coding sequences, indicating the pre¬ sence of specific immunoreaction with the D19 antigen. None of the sera are expected to be immunoreactive with ?-galactosidase.
Anti-D19 antibody present in the sera from the animal immunized with the SJ26/D19 is purified by affinity chromatography (using the D19 ligand, essentially as described above in Example 12 for the anti-yff-galactosidase antibody.
EXAMPLE 14
PCR DETECTION OF N(ABCDE) HEPATITIS SEQUENCES A polymerase chain reaction testing algorithm was devised first to verify exogenicity with respect to several genomic DNAs which could have been inadvertently cloned during library construction, then to test for the presence of the cloned sequence in the cloning source and related specimen materials. Several different types of specimens, including SISPA-amplified nucleic acids and nucleic acids extracted from the primary source, and nucleic acids extracted from related source materials (e . g. , from animal passage studies) , were tested.
A. Amplification from SISPA Uncloned Nucleic Acids
SISPA (Sequence-Independent Single Primer
Amplification) amplified cDNA and DNA were used as templates (Example 2) . Sequence-specific primers designed from selected cloned sequences were used to amplify DNA fragments of interest from the templates. Typically, the templates were the SISPA-amplified samples used in the cloning manipulations. For example, amplification primers 470-20-1-77F (SEQ ID NO:25) and 470-20-1-211R (SEQ ID NO:26) were selected from the clone 470-20-1 sequence (SEQ ID NO:106).
These primers were used in amplification reactions with the SISPA-amplified PNF2161 cDNA as a template.
The identity of the amplified DNA fragments were confirmed by (i) size and (ii) hybridization with the specific oligonucleotide probe 470-20-1-152R (SEQ ID
NO:27), designed based on the 470-20-1 sequence (SEQ ID NO:106). The probe was labelled with 32P using T4 polynucleotide kinase and standard methods for 5'-end labelling. Hybridization to the amplified DNA was then performed using either Southern blot or liquid hybridization (Kumar, et al . , 1989) analyses.
Positive control DNA used in the amplification reactions was previously amplified PCR product whose concentration was estimated by the Hoechst 33258 fluorescence assay, or, alternatively, purified plasmid DNA containing the cloned inserts of interest.
The 470-20-1 specific signal was detected in cDNA amplified by PCR from SISPA-amplified PNF2161. Negative control reactions were nonreactive, and positive control DNA templates were detected. B. Genomic PCR
The term "genomic PCR" refers to testing for the presence of specific sequences in genomic DNA from relevant organisms. For example, a genomic PCR for a Mystax-derived clone would include genomic DNAs as follows:
1. human DNA (1 μg/rxn.)
2. Mystax DNA (0.1-1 μg/rxn.)
3. E. coli (10-100 ng/rxn.) 4. yeast (10-100 ng/rxn.)
Human and Mystax DNAs are tested, as the immediate and ultimate source for the agent. E. coli genomic DNA, as a frequent contaminant of commercial enzyme preparations, is tested. Yeast is also tested, as a ubiquitous organism, whose DNA can contaminate reagents and thus, be cloned.
In addition, a negative control (i.e., buffer or water only) , and positive controls to include approximately 105c/rxn. , are also amplified. Amplification conditions vary, as may be determined for individual sequences, but follow closely the following standard PCR protocol: PCR was performed in reactions containing 10 mM Tris, pH 8.3, 50 mM KCl, 1.75 mM MgCl2, 1.0 uM each primer, 200 uM each dATP, dCTP, and dGTP, and 300 urn dUTP, 2.5 units Taq DNA polymerase, and 0.2 units uracil-N-glycosylase per 100 ul reaction. Cycling was for at least 1 minute at 94°C, followed by 30 to 40 repetitions of denaturation (92-94°C for 15 seconds), annealing (55-56°C for 30 seconds), and extension (72°C for 30 seconds). PCR reagents were assembled, and amplification reactions were constituted, in a specially-designated laboratory maintained free of amplified DNA. As a further barrier to contamination by amplified sequences and thus compromise of the test by "false positives," the PCR was performed with dUTP replacing TTP, in order to render the amplified sequences biochemically distinguishable from native DNA. To enzymatically render unamplifiable any contaminating PCR product, the enzyme uracil-N-glycosylase was included in all genomic PCR reactions. Upon conclusion of thermal cycling, the reactions were held at 72°C to prevent renaturation of uracil-N-glycosylase and possible degradation of amplified U-containing sequences. A Hot Start PCR was performed, using standard techniques ("AMPLIWAX", Perkin-Elmer Biotechnology, Norwalk, CT; alternatively, manual techniques were used) , in order to make the above general protocol more robust for amplification of diverse sequences, which ideally require different amplification conditions for maximal sensitivity and specificity. Detection of amplified DNA was performed by hybridization to specific oligonucleotide probes located internal to the two PCR primer sequences and having no or minimal overlap with the primers. In some cases, direct visualization of electrophoresed PCR products was performed, using ethidium bromide fluorescence, but probe hybridization was in each case also performed, to help ensure discrimination between specific and non-specific amplification products. Hybridization to radiolabelled probes in solution was followed by electrophoresis in 8-15% polyacrylamide gels (as appropriate to the size of the amplified sequence) and autoradiography.
Clone 470-20-1 was tested by genomic PCR, against human, E. coli , and yeast DNAs. No specific sequence was detected in negative control reactions, nor in any genomic DNA which was tested, and 105 copies of DNA/reaction resulted in a readily-detectable signal. This sensitivity (i.e., 105/reaction) is adequate for detection of single-copy human sequences in reactions containing 1 ug total DNA, representing the DNA from approximately 1.5 x 105 cells. All clones discussed herein have tested negative for human DNA, Mystax DNA (mystax derived clones only) , E. coli , and yeast DNA using genomic PCR.
C. Direct Serum PCR
Serum or other cloning source or related source materials were directly tested by PCR using primers from selected cloned sequences. In these experiments, putative N(ABCDE) viral particles (DNA or RNA) were directly precipitated from sera with polyethylene glycol (PEG) , or, in the case of PNF and certain other sera, were pelleted by ultracentrifugation. For DNA isolation, the pelleted materials were digested with proteinase k, followed by phenol/chloroform extraction and ethanol precipitation (Ausubel, et al . ) . For purification of RNA, the pelleted materials were dissolved in guanidinium thiocyanate and extracted by the acid guanidinium phenol technique (Chomczynski, et al . ) . Alternatively, total nucleic acids were prepared by proteinase k/sodium dodecyl sulfate (SDS) digestion and phenol/chloroform extraction, followed by alcohol precipitation.
Isolated DNA was used directly as a template for the PCR. RNA was reverse transcribed using revere transcriptase (Gibco/BRL, Gaithersburg, MD) , and the cDNA product was then used as a template for subsequent PCR amplification.
In the case of 470-20-1, nucleic acid from the equivalent of 50 ul of PNF serum was used as the input template into each RT/PCR or PCR reaction. Primers were designed based on the 470-20-1 sequence, as follows: 470-20-1-77F (SEQ ID NO:25) and 470-20-1-211R (SEQ ID NO:26). Reverse transcription was performed using MMLV-RT (Gibco/BRL) and random hexamers (Promega, Madison, WI) by incubation at room temperature for approximately 10 minutes, 42°C for 15 minutes, and 99°C for 5 minutes, with rapid cooling to 4°C. The synthesized cDNA was amplified directly, without purification, by PCR, in reactions containing 1.75 mM MgCl2, 1 uM each primer, 200 uM each dATP, dCTP, dGTP, and TTP, and 2.5 units Taq DNA polymerase ("AMPLITAQ", Perkin-Elmer, Norwalk, CT) per 100 ul reaction.
Cycling was for at least one minute at 94°C, followed by 40-45 repetitions of denaturation (94°C for 15 seconds for 10 cycles; 92°C for 15 seconds for the succeeding cycles) , annealing (55°C for 30 seconds) , and extension (72°C for 30 seconds) , in the "GENEAMP SYSTEM 9600" thermal cycler (Perkin-Elmer) or comparable cycling conditions in other thermal cyclers (Perkin-Elmer; MJ Research, Watertown, MA) .
Positive controls consisted of previously amplified PCR product whose concentration was estimated using the Hoechst 33258 fluorescence assay, or purified plasmid DNA containing the DNA sequence of interest. In addition, an aliquot of positive control DNA corresponding to approximately 10-100 copies/rxn. was spiked into reactions containing nucleic acids extracted from the cloning source specimen, as a control for the presence of inhibitors of DNA amplification reactions. Each separate extract was tested at least once in this manner. Specific products were detected by hybridization to a specific oligonucleotide probe 470-20-1-152R (SEQ ID NO:27), for confirmation of specificity. Hybridization of 10 ul of PCR product was performed in solution in 20 ul reactions containing approximately 1 x 106 cpm of 32P-labelled 470-20-1-152R. Specific hybrids were detected following electrophoretic separation from unhybridized oligo in polyacrylamide gels, and autoradiography.
In addition to PNF, extracted nucleic acids from several cloning source specimens were reverse transcribed and amplified, using the "serum PCR" protocol sequence. No signal was detected in GB (Mys3721) , Mys29 (Post) , Mys 131 (Post) , Mysl31 (Liver DNA from AT), Mysl31 (Liver RNA from AT), Ful . Hep. DNA (from JL) , NMS, NHS, JS, SCH, MWT or Bonino. The specific signal in PNF serum was reproducibly detected in multiple extracts, with the 470-20-1 specific primers.
EXAMPLE 15 WESTERN BLOT ANALYSIS OF SERA PANELS
The antigens D6, and D19 were screened using panels of sera derived Mystax both prior and subsequent to innoculation with the GB agent and/or PNF. The antigens D6, D19, IA and 17A were screened using panels of human sera derived both from individuals suffering from hepatitis and uninfected controls.
The antigen sj26-pGEX-GLI-l was used as a control. This sample was determined to be at a concentration of 5 mg/ml using multiple assays, and was used as a standard for estimating the concentration of all other antigens tested.
Protein concentrations for all antigens were determined by a combination of determining the OD 280 nm of the protein fractions and by comparison to the protein standard described above. For all antigens 3 protein concentrations in the range of 0.5-3 ug/cm were fractionated using a 12.5% polyacrylimide gel and transferred onto nitrocellulose membrane. Typically 3- 7 cm of membrane containing each protein concentration would be produced. The membrane was then blocked in a solution of "BLOTTO" (150 mM NaCl, 20 mM tris-HCl pH 7.5, 1% normal goat serum, 1% Bovine serum albumin, 1% non-fat dry milk (w/v) and 0.02% sodium azide) at at least 1 hour. The membrane was then dried and cut into 1-2 mm strips. The strips containing different levels of blotted antigen were first rehydrated in TBS (150 mM NaCl; 20 mM Tris HCI, pH 7.5) then incubated overnight with test sera diluted in "BLOTTO" to which was added whole cell lysate of bacteria expressing non¬ recombinant pGEX-GLI at a dilution of 1/20 (v/v) . Along with the strips containing test antigen, one strip containing 3 ug/cm of nonrecombinant pGEX-GLI was also incubated with test sera. Typical sera employed included Mystax 88 for clones D6, and D19 or JFA for IA and 17A, rabbit anti-sj26 sera (diluted 1/1000) , and two control sera derived from blood donors. After overnight incubation at 25°C with gentle rocking, the strips were washed four times with TTBS (TBS plus 0.2- 1.5% "TWEEN 20") and were then incubated with goatn anti-Human IgG (Promega, Madison, WI) conjugated to alkaline phosphatase diluted between 1/2000-1/7500 in "BLOTTO" for 1-2 hours at 25°C with agitation, at which point the strips were washed 4 times with TBS. Bound antibody was detected by incubating the strips in a substrate solution containing BCIP and NBT in pH 9.8 phosphate buffer. Color development was allowed to proceed for -15 minutes at which point color development was halted by 3 washed in distilled H20. The concentration of antigen that gave the strongest signal with the least amount of background or non¬ specific reactivity was determined and employed for all subsequent assays.
Prior to testing with human or Mystax sera 18 X 13 cm nitrocellulose membranes were prepared using the optimum antigen concentrations determined as described above. The antigens were fractionated, and transferred to nitrocellulose as described above. The antigens were fractionated, and transferred to nitrocellulose as described above. Prior to large scale testing, four strips from each of the membranes to be employed were pre-tested by incubation with positive and control data as described above. All membranes used in large-scale testing had to demonstrate immunoreactivity to positive control sera prior to use. Assays with test sera were performed as is described above, with each sera to be tested being incubated with antigen containing pGEX-GLI strips, up to 80 sera were tested in any one assay. Test sera were derived from the following groups of individuals and/or experimental animals.
(i) Serial bleeds from Mystax innoculated with the GB and PNF agents, (ii) sera from control Mystax not innoculated, (iii) sera from individuals who are infected with Hepatitis B virus by virtue of being positive positive for the presence of Hepatitis B surfant antigen, (iv) sera from individuals infected with Hepatitis C virus by virtue of being reactive in a second-generation HCV ELISA assay, (v) sera from blood donors with an above-normal Alanie aminotransferase
(ALT) measurement, (vi) sera from normal blood donors and (vii) sera from individuals suffering from Non-A-E hepatitis.
The antigens D6 and D19 were strongly reactive only with the screening serum Mystax 88. Neither antigen was reactive with pre-inoculate sera from any of the four mystax that were inoculated with GB agent.
Table 11 presents the results obtained from testing the antigens D6, D19, 17A and IA with human sera.
All human sera considered reactive with a particular antigen were tested at least twice against this antigen.
Table 11
REACTIVITY OF D6. D19. IA AND 17A WITH HUMAN SERA
Figure imgf000086_0001
Sera Type D6 D19 IA 17A
Non A-E Hepatitis 7/112 0/23 19/155 1/47
Random Donors 2/54 1/40 1/78 4/36
EXAMPLE 16 IMMUNOSCREENING OF A DEN cDNA LIBRARY A. Construction of DEN cDNA and DNA Libraries. A cDNA library was constructed, essentially as described in Example 2, using RNA isolated from DEN serum (Example 1) .
A DNA library was constructed, essentially as described in Example 3, using DNA isolated from DEN serum. The DEN cDNA source (ATCC 75417) and DNA source (ATCC 75418) libraries were deposited at the American Type Culture Collection, 12301 Parklawn Dr., Rockville, MD 20852.
B. Immunoscreening and Characterization. The cDNA library of DEN in lambda gtll was screened, as described in Example 4, with DEN serum. Two of the exogenous immunoreactive clones were selected and designated DR25-1 and 2DR8. The clones were sequenced. The sequences are presented as SEQ ID NO:51 and SEQ ID NO:103, respectively.
Clones DR25-1 and 2DR8 (i) tested positive for immunoreactivity in secondary plaque screening assays using DEN serum, (ii) was determined to be exogenous to human, mystax, and E. coli genomic DNA, (iii) was non- immunoreactive with normal human sera, and (iv) contained an open reading frame in frame with β- galactosidase.
The sequences of clones DR25-1 and 2DR8 were compared with "GENBANK" and EMBL database sequences at both nucleic acid and amino acid levels. The "GENBANK" search indicated that the sequences are unique as both nucleic acid and amino acid sequences.
EXAMPLE 17
IMMUNOSCREENING OF GB cDNA AND DNA LIBRARIES A. Construction of GB cDNA and DNA Libraries. Two cDNA libraries were constructed, essentially as described in Example 2, using RNA isolated from My 620 and My 670 sera (Example 2) and adding E/A' linkers (SEQ ID NO:22 and SEQ ID NO:49) to both ends of the molecules.
Two DNA libraries were constructed, essentially as described in Example 3, using DNA isolated from My 620 and My 670 sera.
The cDNA and DNA source libraries were deposited at Genelabs Technologies, Incorporated, 505 Penobscot Drive, Redwood City, CA 94063.
B. Immunoscreening and Characterization. Twenty one clones (i) tested positive for immunoreactivity in the plaque screening assays using the mixture of convalescent sera from animals inoculated with GB agent, (ii) were determined to be exogenous to human, mystax, and E. coli genomic DNA, and (iii) were non-immunoreactive with pre-inoculum and/or normal sera. Further, the insert sequences were determined to be novel (i.e., they did not have significant sequence homology to any GENBANK sequences) and each contained an open reading frame continuous with the j8-galactosidase coding sequence of the lambda gtll vector.
Clones whose designations begin with "468" and "472" were isolated from cDNA libraries made from My 670 serum. Clones whose names begin with "474" or "486" were isolated from cDNA and DNA libraries, respectively, made from My 620 serum. Clones whose names begin with "475" were isolated from DNA libraries made from My 670 serum.
These clones are identified by SEQ ID NO:11 through SEQ ID NO:16, SEQ ID NO:39 through SEQ ID NO:43, and SEQ ID NO:74 through SEQ ID NO:83.
While the invention has been described with reference to specific methods and embodiments, it will be appreciated that various modifications and changes may be made without departing from the invention.
SEQUENCE LISTING (1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Genelabs Technologies, Inc.
(B) STREET: 505 Penobscot Drive
(C) CITY: Redwood City
(D) STATE: CA
(E) COUNTRY: USA
(F) POSTAL CODE: 94063
(i) APPLICANT:
(A) NAME: United States of America, The, as represented by the Secretary of the Department of Health and Human Services
(C) CITY: Washington
(D) STATE: DC
(E) COUNTRY: USA
(F) POSTAL CODE: 20231
(ii) TITLE OF INVENTION: Non-A/Non-B/Non-C/Non-D/Non-E Hepatitis
Agents and Molecular Cloning Thereof
(iii) NUMBER OF SEQUENCES: 106
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Dehlinger & Associates
(B) STREET: 350 Cambridge Avenue, Suite 250
(C) CITY: Palo Alto
(D) STATE: CA
(E) COUNTRY: USA
(F) ZIP: 94306
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentin Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION: 89
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/246,986
(B) FILING DATE: 20-MAY-1994
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Fabian, Gary R.
(B) REGISTRATION NUMBER: 33,875
(C) REFERENCE/DOCKET NUMBER: 4600-0201.49
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (415) 324-0880
(B) TELEFAX: (415) 324-0960
(2) INFORMATION FOR SEQ ID Nθ:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 234 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: My 131 Clone 427-7-1
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
GAATTCGCGG CCGCTCGACA CAGGAGACAG AGGGATACAC ACAGGGGCAC GGAAATAAAT 60
ACTGGACAGA AAAGGATACA CACAGGAGAG TGGAGGAAAC ACAAAGAAGA AAGAAGCATA 120
CACGGAGGAG AAAAAAATAC ACACAAAAGA GGAAGATACA CACAGAAAGG AAAAGGATAC 180
ATAGAGGAAA TGGAAATACA CACAGGCGAC ATGGGCCCGA GCGGCCGCGA ATTC 234
(2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 133 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: My 131 Clone 430-2-14
(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:2:
GAATTCCGGG CCCTGCCACG ACAGCTGCCT TCCTGCACAA AATGTCAAAG CTCTTGATGG 60
CCTTAGCTTT GCCTTTCTGG AGAATGTTCT AGACTGGCAT GCTCCCGGTT GTATAGCGAG 120
CGGCCGCGAA TTC 133
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 165 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: My 131 Clone 430-3-4
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GAATTCGCGG CCGCTCGAAA TACACACAGG AGCGAGGGTG ATGCACACAG GAGTAAAAAA 60
AATACACAAA GGAAAGACGA GAATACACAC AGGAGAGAGA AAAATACACA GAGGAGAGGG 120
GCGTACACAC AGTAGAGAAA AGGATACACG AGCGGCCGCG AATTC 165
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 216 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: My 131 Clone 430-4-8
(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:4:
GAATTCGCGG CCGCTCGGAT CGGCAAGACA GTGGAGGCAT ACATAGACGA TGTGGTCATT 60
AAAACCAGAC ACGTCGACTC CTTAATAGAC GACTTGAGGC TCACGGTCGA CAATATCCGA 120
ACATACGACA TTAAGCTCAA TCCGGAAATA TGCGTTTTCG GCGTACCCGC CGAAAAGCTC 180
CTGGGCTTCA TCGTCTCCAC GAGCGGCCGC GAATTC 216
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 204 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: My 131 Clone 428-2-3
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
GAATTCGCGG CCGCTCGTGC AGCTATTCTT ACTGGAGAAG CAGCAGCGAG TGTGAACATT 60
TCCGCAGGGG ATGGGAAGAA GGAAAAGCAG TCACGGGGAC ATTCCTCACC CCAGGGGACC 120
AGTGATGCTC CATTTAACGG CACTAGCAGA TCGAGTCCCA TTTCTGCTTT GAACAGAATG 180
TTCGAGCCGA GCGGCCGCGA ATTC 204
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 150 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: My 131 Clone 428-3-1
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
GAATTCGCGG CCGCTCGGGG GGAAGGGAAC CACCAAGGTT CTGCTGACTC TCCTCTAAAT 60
GAATTTTTAT TGTTTTGTTT TTTGGTCAGT TATGAGGAGT CAAATCCAAA GGATCCAGCG 120
GCAGTGACAG AATCGAGCGG CCGCGAATTC 150 (2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 135 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: My 131 Clone 428-7-3
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
GAATTCGCGG CCGCTCGGGC AAGGCAATGT AAGGCAAATC CAGTGGAGGC AAATCCAAAC 60
AAAACAATTC CAGGAAAGGT AAGGCAAGAC AATGCAGGCA AAGGCAATTC CAGGCAAGAC 120
GAGCGCCGCG AATTC 135
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: Sispa C linker (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
CGAATTCGGC CAAGTCGGCC 20
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 267 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: My 131 Clone 430-2-1
(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:9:
GAATTCGCGG CCGCTCGGGA GAGAAGGGGA AAGCACACAG GAGAGTGGAA AGATACACAC 60
AGAAGAGAGA AGGATACACA CAAAAGAGAG GAGGATACGC ACAAGAAGAA AAGTATACAC 120
ACAGGAAAGT GTGATACACA CATGAGAGAA GGGTGTAAAT ACAGGAGAGA GGAGGATACA 180
CACAAAAGAG TGGTGGGATA CACAAGAAAA GGAGAAGGAT ACACACAGTA GAGAGTAGAA 240
TACATAAAGG CGAGCGGCCG CGAATTC 267
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 351 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: My 131 Clone 430-4-1
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
GAATTCGCGG CCGCTCGCAA CAGCCAACAT TCAAAGCAAC TGATTTTCCA AAAGTCCAGC 60
AAACAGTTGA TGAATTTAGC AAATTCATAC TTTTTTTTCT GCGTGCCCCT ACCTATTACC 120
CTGCCTCTGC CTCTACCTCT TGAATATTCA TCTCCAAATT TCTATCAAAA TGACCAATGG 180
AATTGAACAC TAGTCAATGA ATGGAGAATT TACCAATGTC ACTAACTTTA TTCAGATCCC 240
CGCAGTAGTT ATGGTAAAGT GGGTGGGTTA AGTGTTCTAA CGGAATATTT TAATTACTTA 300
TTTCATCCAA TAGCACCCTG ATATCTTATA AACCCGAGCG GCCGCGAATT C 351
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 80 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: GB Clone 472-2-1
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
GAA TTC GTT ACA CAG GAA AGA GAA GGT ATA CTC ACG GGA GAA AAG AAA 48 ATA CAC AAA GAA GGG CCA CGC CTA GGG AAT TC 80 (2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 153 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: GB Clone 474-19-3
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
GAATTCCCTA GGCGTGGCAC ACAGGAGAGA GGAGGATACA CACAGGATAA GGGGATACAC 60
ACAGGAAAGA GGGATACACA AAGGAGAAAA GATACACACA GGAGAGAGCG GCATACGCAC 120
ATTAAAGGAT GATAAACAAA GGAGAACGAA TTC 153
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 202 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: GB Clone 474-6-3 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
GAA TTC GTT ATA GGG GAA ACA GAA GGC CAA CAA GTT GGA GGA AAT CTT 48
GGA GAG GAA GAA AGT GTT AGG GAA GGA GAG AGG GGC TTC TTG TCA AGC 96
TCT AAT GTG AGG GAG TCG TAA GGA GGC TGA GAA TTG ACC ATT TAG TTG 144
ACA AGA CAG TTT GCT GGT GAC CGT GTT TCC CCT ATA AAT CCC CAC GCC 192
TAG GGA ATT C 202
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 231 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: GB Clone 475-12-3
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
GAA TTC GTT CGC ACC GAA GGT CAC GAC ACC CTT GAT ATC GAC CAG CTT 48
GCC CTG GGC GCT CAG CAC GGC CGA GGA ACT CAG CGT GCT CAC GCC GCC 96
CTG GTA GGA CGG ACG CAG GTA GCC CTC GCC GGA TGC GCC GTT GTC GGT 144
GAT GCC CGC CAG CAG GAC GTA GGG CGC CGA CAG CGA CAC GCT GTG GAT 192
CGG ATC CAT GCC GGC CGC GAC CCC ACG CCT AGG GAA TTC 231
(2) INFORMATION FOR SEQ ID NO:15: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 216 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: GB Clone 475-16-4
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
GAA TTC GTT AGA GAA AAG ACA TCC CTC TCC ATT TTT GGA GCA AAA CAT 48
CCT CAA AGC AGT GAA GTT AGA CCC ATT CAG AGG CTC CTG GAG GGA TTC 96
CCA AGG AAA CTG TCA GTG GGA ATG TGT GGA GCA AAA CAT CCT CAA AGC 144
AGT GAA GTT AGA CCC ATT CAG AGG CTC CTG GAG GGA TTC CCA AGG AAA 192
CTG TCA GCC ACG CCT AGG GAA TTC 216
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 123 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: GB Clone 475-3-3 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
GAA TTC TCT AAG TAC TAC TAC AAT CAA GAA ATT GAT CTA ACC AGT CTA 48
CTA GAC CTA GAT GAT CAA GAT GGT GAA AAT GCA TAC TGT AAG TAT CCA 96
AAA AGA TTT GAC AAC ATC AAT GAA TTC 123
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 138 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: My 190 Clone D19
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
GAATTCGCGG CCGCTCGGGC ATTATTTTCA TTCGAGGACG TGCCTTCCTC TTTGCCTCCT 60
GCTGTCACTA ACATTTTGCA GGAGTTCGCT GACGTTTTTC CACAAGACGT GCCACCGGGA 120
TCGAGCGGCC GCGAATTC 138
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 181 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: My 190 Clone D20
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
GAATTCGCGG CCGCTCGCAA GCAAGATTCC ACCAGCAAAA AGAATCCATC AGGACCTAGT 60
AAACATTGAG GGGCCAGTAG AAGCACTCTC CTACCAGGCT CATCCTGAAA CCCCACTCCC 120
ACATCTAGGA ATATCCAGGT AGCTGTGTGC CAGCCAGCAA GGGGGCGAGC GGCGCGAATT 180
C 181
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: Sispa D linker
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
GGCCGACTTG GCCGAATTCG TT 22
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 304 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: My 190 Clone D30
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
GAATTCGCGG CCGCTCGCGG CGAAGCACCG CCGGACGGAG GATCCTCGTC GCCGCCGTGT 60
GGAGGGAAGC CACCATCCTC GGAGCCCTGT GAACGAGCAC GAATCAGTCG CCCAAGACTG 120
GAAAAATCAA ACCGGGATGG AACCTAACTT GCGGGTCGGA ACTACTGCAG AAGTGGTGGC 180
TCTGGTGTCA GCAACTGGCA CGGCAGACGA CCTCGGACCT CGTGGCACTC TGGCAAAGGC 240
ATGAGTGGTA GTCCTGCCTC TCCTCTCCTG CATATCATCC TGGAGCTCGA GCGGCCGCGA 300
ATTC 304
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 135 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: My 190 Clone D44-2
(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:21:
GAATTCGCGG CCGCTCGGAT CTTTCGGGCA GGCCCGGTTG ATGTGCTTGA AATCAATGCA 60
CATTCGGAGC GACTTGTCCT TCTTAGGAAC CATGACGACA TTAGCGAGCC ACTCGGAGCG 120
AGCGGCCGCG AATTC 135
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: Sispa E linker
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
CGAATTCCCT AGGCGTGG 18
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 311 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: My 190 Clone D48
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
GAATTCGCGG CCGCTCGCTC GCAGGCGGCG GCAGGGGAGG GGAGAAGGGG AGGAGGAGGG 60
GTCCCGGCGG CGGTGGCGCT AGGGTTTCTC CCATGACGCG CGTCACGGGA GGAGGACGGA 120
GGGGATTGTG TTCGAATTAA TTCGCGGCCG CTCGGAACGC GGCATCGAGT GCAACCCAGT 180
GAAGATCAAG GCCATAGAGA GAACGGAGAT TCCTACCAAG CTGCGAGACA TCCAAAAGTT 240
TACCGGGTGC CTAGCCTCCC TGAACCGCTT CATCAGCCAG GTAGGAGAGA AGGCCGAGCG 300
GCCGCGAATT C 311
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 192 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: My 190 Clone D5
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
GAATTCGCGA CCGCTCGGGA AGAAAGCAAG GGAAAGAATG CCCCTCATCC CTCTTATCTC 60 TCATCTCCTG CCAGGGCTTG CCATTGGCTT AACCCAGTGG ACAGAGAGCC TGAATATGCA 120
GTCCATGTAA ACCAACCTCC TGGGGACCTG TGGACAAGGG CAAAGTGGGC TCTGGCGAGC 180
GGCCGCGAAT TC 192
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: Primer 470-20-1-77F
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
CTCTTTGTGT AGTAGCCGAG AGAT 24
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: Primer 470-20-1-211R
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
CGAATGAGTC AGAGGACGGG GTAT 24
(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: Primer 470-20-1-152R
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
TCGGTTACTG AGAGCAGCTC AGATGAG 27
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 290 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: My 190 Clone D6-1
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
GAATTCGCGG CCGCTCGAAT ATGGTCTGGC GCCGAAGTCG ATGTTGATGC AGGGCATCGG 60
CGTGATGCGG TAAAGGCGTG GCAAAGCCTG AATTAATTCG CGGCCGCTCG CCCGTCCGTG 120
CAAAGTCAAA GGGCTAGATC CCGTGGTCAA ACGCTCCAGG GTTAGCCGGG ACGGGGGCCC 180
TGGGGGGGAA GCAATGCCAT CGGAGCGTCG TACCGGGCCC TCATACATGC GGGGTGGTGT 240
TGTGGCATGT CTTAAGAGGC GACACGCGTC TCCCGAGCGG CCGCGAATTC 290
(2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 185 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: My 190 Clone D64-1
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
GAATTCGCGG CCGCTCGGAA GGGTTTAGCA GACAAAGACA GCTGCCTCCC TAGACAGCCC 60
AAACAGGAAC TTCTCCAGAC AAGGACAGCC CAGCCTGACA GACCAGGGAA GGTTGGAGCA 120
GGGAGGCTCA GGAGCAAAGA CTTGAGTAAG AGTTAGGAGT TCCTGAGACG AGCGGCCGCG 180
AATTC 185 (2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: Primer 11F
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
CACATGGCTG AATATCGACG 20
(2) INFORMATION FOR SEQ ID NO:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: Primer 11R
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:
GGCAGACATG GCCTGCCCGG 20 (2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 145 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: My 190 Clone D24
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
GAATTCGCGG CCGCTCGGCC GGACTCCTCG TCATTGAAGC CAGGTTCAGG GGCTACTGAG 60
GGAGTCCTGG ACTAAGGGGT CCTCGGGCGT CCGGCCTGTT ACTCATTGGG CCAGACTGAT 120
GGGCTATGCG AGCGGCCGCG AATTC 145
(2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1062 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: clone R27
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: GAAATTCGCG GCCGCTCGGG AGAAACAAGT GTGAGCAGCT GGTTTGCCGA GGGGAAGACA 60
GTGAATGGGG CTGGCTCTCA GGTGTGCGCA GAAGCCGGCC TTGGCACCGG GTTTGAGGAA 120
AGACAGGCTT ATTTTGGGCC CCACACAAGG AGCCAGGATC CGGCTCACAT CCGTCTCCCC 180
ATCCTGGCCG AGCGGCCGCG AATTAATTCG CGGCCGCTCG CAGGTCAGGG ATCCATGGGT 240
TTTTGCAACA GAAGCCGTCC ATGCTCTTCC TCTGGAAGGT GTAGCTTTTG CTGTCAGGGT 300
GGCTGCTTTA AATTCTCCCG GGTGATGGTC TGGATCTCGC GGTCAGCAAT TGGGGAAAGG 360
TGGGAAAGTG TGTTACTGCT CATTTCCGGG GCCTTCTAGT GCCTCAGGCT GGGCTGTTCC 420
AGAAGGGCCC ATTCTTTCCT CAGACCTTGG TCCTTCCCGG GGAATAGTCC TTCTCCCGTT 480
CTCAGCCAAG TGGTCCACAT AAGTGGCCCC ATCCCTTCAA GAGTAGGCCC TGAACGGCTA 540
ATGGACCGAG CGGCCGCGAA TTAATTCGCG GCCGCTCGCT CAGGTTGCTG ATAAATGTTT 600
ACTCATAGGC CAGGCCTGCA CAGGCCACAG ACAAGGAGAG GAAGCAGTTT GTGGCTGGAG 660
AGTCGGACAG TCCCACACCC GCGCTCTGCT GTGCTCTTGG CCAGGCAGCC TGGGAGAGGG 720
TGGCCCGAGC GGCCGCGAAT AATTCGCGGC CGCTCGCTAG AAGACCCCGT CGTCTCAGCC 780
CAAAATCTGC TGAAACGGAT AAGTAACTTC AGCAAAGTCT CAGGATACAA AATCAATGTG 840
CAAAAGTCAC AAGCATTCCT ATACCTCAAT AACAGACTTG AAGAGTGCCA AATCAAGAAC 900
GAACTGCCAT TCACAATTGC CGAGCGGCCG CGAATTAATT CAGATGTCAG GACTGGAAAG 960
AGCACAGTAG AAGGATGTCT TAGGTCTTCC ACTCTACAGA AGAAAAAGAT TCAGACCCAG 1020
TGTTTTCCAT CCCACCCAGC AACAGCGAGC GGCCGCGAAT TC 1062
(2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: Primer 11F (JF)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34;
GGCGACGACT CCTGGAGCC 19
(2) INFORMATION FOR SEQ ID NO:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: Primer 11R (JF)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:35ι
CCAACTGGTA ATGGTAGCG 19
(2) INFORMATION FOR SEQ ID NO:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 624 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: JFA Clone 17A21
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:
GAATTCGCGG CCGCTCGGCC AGAAACGGCC TTGGAAAAGA AATTACGCCA AAACCAAGCA 60
GCGGTTGAAA AACGCAACGC GTTGGCCAAG AAAAATAGTG ACACCAGTGC GGCTAAAAAA 120
GGGCAGTCGA CGCACTTGTC ACCAGAACAG CAAGCCCTTG TTAAAAATTA TGGCTTACAG 180
CCAGCCCAGT ATCTCGCTTT CCAAGCCATG CCATTTCAGG CTTTTGGTGA TTCGGTCATG 240
TTAGATGCTG CGCCTTATCT ACAAGAAGTT AATCCACATA TGGTGGTGGA TGCGGCGGTT 300
GGTCGACAAC CTTATCAAAC GCCAAAAATC ATGGCGCAAG CCGCAGCCGC ACAACCATTA 360
GCAGATAATC TGCTCATTGG CTTAGGGACA AATGGGACGA TTAAGAAGCA AGACTTAGAT 420
CAAATTATGG CAATTGCCGG TAAAAAGCGT CAGGTCTATT GGATGAATAA CTTTGTGCAG 480
TCTCGTCCTT GGCAGGATAG TAACAATCAA TTGTTACAAA CCGCGCAGAA AACGTATAAG 540
AATTTGCACG TGGTTGATTG GTACGCAGTT GCCAAGCAAC ACGGTGACTG GTTTGCCGAT 600
GATGGGGCGA GCGGCCGCGA ATTC 624
(2) INFORMATION FOR SEQ ID NO:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 501 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: JFA Clone 1A2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:
GAATTCGCGG CCGCTCGCGA ACTCGTTGAG AGCCTCGACC ACCAGTGCGC ACTCATCGTT 60
ACTGATCCCC GTCGTCTTGA CGAGATCGTC GGCCTTCAAG CCCAGTCGGA CCGCCCCGTC 120
GTCGTCCTGC TCGCCACCGA CCCTGACGAC GCTCCTGACA AGCTTGCGGA AATTGTTGAC 180
GTTGTCGTCA CCCCTAACCC CCAGACCGAA CCTTGGGAAC ACCTCGGCGT CCCGGTGCAT 240
TCCATCGGCG TCCCGGCGAC TGGCGGCGGT CCGGCCCCTG GCGACCGCCC CAACCGAATT 300
GCCCTACTTG GCCCGTGCAC CGATGAGCAG CTTGCCGACG CTCTCCTCGC CTTCGACCAC 360
GCCTGCCAAG TTGTGCCCGG CTGGTCCTTG GAGATCTGCC TTGACGATGA ATCCAGAGCC 420
CGCTCGGCAG TGGCAGCCCG CCGCGACGTC GGTATTCACC CAGCTGGATC CGAAAACACG 480
GTTACGAGCG GCCGCGAATT C 501
(2) INFORMATION FOR SEQ ID NO:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 612 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: JFA Clone 4B11 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:
GAATTCGCGG CCGCTCGTGG ACGCGGTCAT CGAGTTCGAG CGCCAGGCGC TGCACGACTC 60
GACGTCCGAG CGCGCGGCGG TCTGAGGAGC GGTGCCATGA AGGACATCAC CCTCTTTCCC 120
GCCGACATCG CCGCAATGAC CGTCGGCCAA CTGGCCGCGT TGCCCGCCGT GCAGAAGGCC 180
GAGATCGACA AGAACCGCGA GCGGCCGCGA ATTCGCGGCC GCTCGCCCAA AATATTTGCT 240
CGTAATTTGA GATCTCTGCA AAACAATGCA CCTCCTGGCA AAAACATCGA TGTCAATTGT 300
TTGAACGTCA ATTCTTGTTC GTTGTCCGCA AGCCCAAGCT CACAAATTAA CATGGCTTGT 360
AATGGAAACA AGCAAGATCT TCCCATACCG TTTCCCCTGC ACGAGCGGCC GCGAATTCGC 420
GGCCGCTCGC CCATCTCTTA TAAGGACTCA AATCTTTACG TTAACGGCAA GCAAGTTAAC 480
CAAGATTATA TTGGGATTAA TGAACGGACC GAAGGCACAG AGATGTCATT TGGTAAGAAC 540
TGGTCATTAG CGAGTCTGTC AGCGAGTGAT TTGTGGCAAA AGAAGGATCG TAACACGAGC 600
GGCCGCGAAT TC 612
(2) INFORMATION FOR SEQ ID NO:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 333 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: GB Clone 478-4-1 114 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:
GAATTCGTTC GCCTGCTCGG CGAGATACGC GCGCGACAGG ATCAGCGTGA CGCCGGAGCC 60
GAGCAGCGCC AGGATTTTCA GGAACCGCGC AAAATCATCG ACAATAAAGC TGCCGCCGAA 120
GGTCGTGAGC TTGCCGCCGG GCAGCCACAG CACCAGGGCA CCCGTGATAA CGAGGAAAGC 180
AACCGCAAGA CCGGTCACGA GTTGTGTCGT CTGCCCGCTG CGAAACGCGC CCAGCATCAG 240
CAGCGCCATG GCGCCGGCGG CCAGCGCCAG TTCCGGCAGC ACGGGGAGCA GCGAATATCC 300
TGCGAAAGTC ATAAGCCCAC GCCTAGGGAA TTC 333
(2) INFORMATION FOR SEQ ID NO:40:
(i) SEQUENCE CHARACTERISTICS:.
(A) LENGTH: 121 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: GB Clone 486-17-5
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 0:
GAATTCCCTA GGCGTGGGTC CAATTTATTT AGATGCAATA GTTGTGATTT GGAAATCACT 60
AAAGAGGATT TGATTAATTC AAATCAAGAA AACATAAATG CAAATCTCCA TGAAATTGAA 120
C 121
(2) INFORMATION FOR SEQ ID NO:41: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 213 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: GB Clone 486-19-20
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:
GAA TTC GTT GGT AAT AAG GTG GAG ATA GGT AAG AGT TGG CCT AAT AGG 48
GGC AAG CTA TTG AAG TTT GTT TAT GGA AAT GAG GAG GAG ACT AGT ACA 96
GGG GAT TTG GAG GCA AGG CGT AGA GAG AAG TGT CAG TCT TTA ATG GAG 144
GGC CCG CTT TGG TAG GGT AGC AGG GGC TGG AAC CCA GTG GCA GTG ACC 192
TGG GCC ACG CCT AGG GAA TTC 213
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 183 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: GB Clone 486-19-21 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:
GAA TTC GTT CTC CCA TTT CTG CCC AGC CAC ATC CTA TCA TGG TCA GCA 48
GCC AGC CCC AGC GCC ACC TTG GTG AAG CCT TCA GGG AGA CCT TGC CAT 96
CCC AGC AAG GGG CAC AGT AAG GAT GCC ACA GTC ACC CCA CCC CAC CCT 144
CAG GGC CAC CTC ATC TGG CAC CCC ACG CCT AGG GAA TTC 183
(2) INFORMATION FOR SEQ ID NO:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 80 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: GB Clone 486-6-4
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:
GAA TTC CCT AGG CGT GGC CAG AAT ACA ACC AAA AAG AGA AAG AAA GGA 48
CCC CAA AGC AAT ACT AAT AAT CCA ACG AAT TC 80
(2) INFORMATION FOR SEQ ID NO:44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: SISPA primer, top strand Linker AB
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:
GGAATTCGCG GCCGCTCG 18
(2) INFORMATION FOR SEQ ID NO:45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: Linker AB, bottom strand
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:
CGAGCGGCCG CGAATTCCTT 20
(2) INFORMATION FOR SEQ ID NO:46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 745 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: clone D12-3 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:
GAATTCGCGG CCGCTCGCCA TGGAGATCAC CGCCGCAAAA GGAAGAAGGA GGGGTGGAGG 60
AGGGGAGGAT CTAATGAGGC GACGAAATGG GGAGACGGTG GCGGCGCAGA TCGGAAGCGG 120
AAGCCGGAAA CCCTAGCAAG GGGGAGAGAG AGCCGCCGAG CGGCCGCGAA TTAATTCGCG 180
GCCGCTCGGC TTTCCTTCTG TTGATCTACG CTTCGCCATC CCCACGCTTC GCCCCCATGG 240
CCGGGACTGC TCTTCCGATT GCGATGATAG ACGCCCGCGG CGGGGCCAGG GGGCTCCGAG 300
CCTTGGCCCC TGCCTTGCGG CGGGTGCAGT GGCCACCCAG GTTCAAGCCA GAAATGCAGA 360
GCCGGACATG ATTCTTGTGT TCAAGATCTA CTTTGAAGTA TTAGGAGAAG GACCCCGCCT 420
TGCAATGCTG AAGACAATCT GCGCGTCGGA CTCATCGTCA TTGAAGCCTG GTTCAGGGGC 480
TACTGAGGGA GTCCTAGATT AAGGGGTCCT CGGATAGCCG GCGAGCGGCC GCGAATTAAT 540
TCGCGGCCGC TCGTTAGAAT CGTCCGGATC AGTGTCAAAG CTTGCATCGA CGTAACCATT 600
TACGACTAGC TCTTTGTCAC CTCCATATAC GAGAAACATA TCCTTAGTCC TTTTCAGGTA 660
TTTCAGGATG TTCTTGACCG TTGTCCAGTG ATCCACTCCT GGATTACTTT GGTACCTACC 720
TGCCAAGCCG AGCGGCCGCG AATTC 745
(2) INFORMATION FOR SEQ ID NO:47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 201 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: clone D13 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:
GAATTCGCGG CCGCTCGGCA GAATGCAGTT GGCAGTTTTC CCAACACTTA CAGAACGAGC 60
TTGGTTGCAC TGCCCTCCAA AATGTGGACA CCAGGCTTCT CAGAGTCTGA GCCTCAGACT 120
CCTCTCAGCT CAGAGACATT AGCACCAGCA AGTGGCATCC GCTCTTCAGA GGCCCAAGCT 180
CCAGCGAGCG GCCGCGAATT C 201
(2) INFORMATION FOR SEQ ID NO:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 519 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: clone D31-2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:
GAATTCGCGG CCGCTCGGGA GAAGCAGAGG AACCAGGGAA GTATGGCGCG GGCGATGGAA 60
GAATAACTGT GGAGGTTGCG GTGGCCGCGT AGCCAGGCGG ATTCCAAGGC TGCGTGGGGA 120
AGCGGGGGCG CCCACGTCCA ATCAACCGCC ACGCGAGCGG CCGCGAATTA ATTCGCGGCC 180
GCTCGCCTGT TAAGGACGTG CTAGAGAGCT CCACCGGCAA GAGCAAGAAG TCCAAGGTTG 240
CACCACCAGA AACTAAGAAG GTCTCCGTCA AGGAGGACGG CACGGGCAGG GCTTTCACTA 300
TAAGCTCCAC GCTCGACAGC AAATAGGAAA GCCGAGCGGC CGCGAATTAA TTCGCGGCCG 360
CTCGGCATCA GATCCAGCAG CCGCCCCAGT CGGTCTGCCT CTGCATCGAC CAGGCCCAAG 420
GCCCAGGGTG AGCAGCCCCC TGGCCTTTCC TCTATTGGGC TAGTCAATTT CGGCCCGTAT 480 AGTGTTTTTT TTCCTGCCTG CGCGAGCGGC CGCGAATTC 519
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(Vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: Sispa A' linker
( i) SEQUENCE DESCRIPTION: SEQ ID NO:49:
CCACGCCTAG GGAATTCGTT 20
(2) INFORMATION FOR SEQ ID NO:50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 579 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: clone D76
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:
GAATTCGCGG CCGCTCGCGA GCCTCCTCCT CGGCAGCTAG AACGAATGTC AGCTCGATCC 60 CCATCTGGGC AGGAGTCTAT GGAGACGCCA GATCCATGCT CTCTGGCTGC CGGAGAGTAT 120
GAACAACCAG AGCATGGTTG GCCGGGCGCC GATTCCGAGC TGCCGAGCGG CCGCGAATTC 180
AATTCGCGGC CGCTCGCGGT TAGGATCCCA TGAACCAGGA GCGCCAGCAC CGGTTCCTCG 240
ATCTTCCTAC TGGAGGCATG GTCATGGCTT TCATTCATTC ATTTCATTCT TAGTTCTGTT 300
GTGGAATCGA ACCGAGCGGC CGCGAATTAA TTCGCGGCCG CTCGCGAGCG GCCTCAAAGC 360
AACAGGCAGG AACCCTGCAG CTGTGATAGC GACGCAGGGC TTGCTGATAG CGTGCAGCAC 420
GCGTAGCAGC CCGAAGATGG TTCTCCTCGA GTAGCACGGC CGAGCGGCCG CGAATTAATT 480
CGCGGCCGCT CGGCCGGGGG TGGTGCGGCC ATGGCAAGCT GCTGAGCTAC GGGGACCACA 540
ATCGTCTCCT TCTTCTTCTT CACGAGCGGC CGCGAATTC 579
(2) INFORMATION FOR SEQ ID NO:51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 231 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: clone DR25-1
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:
GAATTCGCGG CCGCTCGGTA TCGTCCCGGC GCGGTGGATA TCACACCGGA ATCGCTGAAA 60
TTCTGGATTG ACTATATCTC CGGAGGGACA GGGCGCTTCA TTTCCAAAAC CACGGATGCG 120
GCGGTGAAAT CGCTGAATGG TATTGATATA CCGGAACAGC AGGTGCCCTT CCTGGGGAAA 180
ATTTCGGGTG AGGTGATGCC GTATGCAGAC CAGCCGAGCG GCCGCGAATT C 231 (2) INFORMATION FOR SEQ ID NO:52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: Probe 1, 17A-312F
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:
TATCAAACGC CAAAAATCAT GGCGCAAGCC GCAGCCGC 38
(2) INFORMATION FOR SEQ ID NO:53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: Probe 2, WT54-592F
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:
CATCAAGGAC CGTTGGGCGA TCGCCAGTAT 30
(2) INFORMATION FOR SEQ ID NO:54:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: oligonucleotide primer 428-2-3F
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:
CGCCATGGCA GCTATTCTTA CTGGAGAA 28
(2) INFORMATION FOR SEQ ID NO:55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: oligonucleotide primer 428-2-3R
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:
CGGGATCCCT ATTATCTGTT CAAAGCAGAA ATGGG 35
(2) INFORMATION FOR SEQ ID NO:56:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: oligonucleotide primer 17A-57F, 5'
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:
GGCCATGGCG GCGGTTGAAA AACGC 25
(2) INFORMATION FOR SEQ ID NO:57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single.
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: oligonucleotide primer 17A-602R, 3'
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:
CCATCATCGG CAAACCAG 18
(2) INFORMATION FOR SEQ ID NO:58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 340 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: clone WT54
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:
GCGGCCGCTC GCGATTAAGA AGCAAGACTT AGATCAAATT ATGGCAATTG CCGGTAAAAA 60
GCGTCAGGTC TATTGGATGA ATAACTTTGT GCAGTCTCGT CCTTGGCAGG ATAGTAACAA 120
TCAATTGTTA CAAACCGCGC AGAAAACGTA TAAGAATTTG CACGTGGTTG ATTGGTACGC 180
AGTTGCCAAG CAACACGGTG ACTGGTTTGC CGATGATGGG GTACATCAAG GACCGTTGGG 240
CGATCGCCAG TATGTTCGGT TACTCGTTGA AACGGTGGGC CGCGTGTCAG GTGTTAAGTA 300
AACTACCGTC AGGTAGTTTT TTTATTTATT TGGGGTCTAG 340
(2) INFORMATION FOR SEQ ID NO:59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: oligonucleotide primer WT54-590F
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:
TACATCAAGG ACCGTTGGGC GATCG 25
(2) INFORMATION FOR SEQ ID NO:60:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: oligonucleotide primer WT54-684R
( i) SEQUENCE DESCRIPTION: SEQ ID NO:60:
CTACCTGACG GTAGTTTACT TAACAC 26
(2) INFORMATION FOR SEQ ID NO:61:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: oligonucleotide primer, 17A-215F
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:
CCAAGCCATG CCATTTCAGG 20
(2) INFORMATION FOR SEQ ID NO:62:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: oligonucleotide primer, 17A-258F
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:
GGCCATGGAT GCTGCGCCTT ATCTACAAG 29
(2) INFORMATION FOR SEQ ID NO:63:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: oligonucleotide primer, WT54-647R
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:
CCCACCGTTT CAACGAGTAA CCGAAC 26
(2) INFORMATION FOR SEQ ID NO:64:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: Primer KL-1
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:
GCAGGATCCG AATTCGCATC TAGAGAT 27
(2) INFORMATION FOR SEQ ID NO:65:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: Primer KL-2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:
ATCTCTAGAT GCGAATTCGG ATCCTGCGA 29
(2) INFORMATION FOR SEQ ID NO:66:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: oligonucleotide primer D6-NF
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:
CGCGCCATGG TATGGTCTGG CGCCGAAG 28
(2) INFORMATION FOR SEQ ID NO:67:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: oligonucleotide primer D6-BR
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:
CGCGGGATCC CTATTACGTT TGACCACGGG ATCTAG 36
(2) INFORMATION FOR SEQ ID NO:68:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: oligonucleotide primer D19-NF (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:
CGCGCCATGG CATTATTTTC ATTCGAGGAC G 31
(2) INFORMATION FOR SEQ ID NO:69:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: oligonucleotide primer D19-BR
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:
CGCGGGATCC CTATTATCCC GGTGGCACGT CTTGTG 36
(2) INFORMATION FOR SEQ ID NO:70:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: oligonucleotide primer 17A-NF, 5'
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:
CGGCCATGGA ATTCCCAGAA ACGGCCTTGG 30 (2) INFORMATION FOR SEQ ID NO:71:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: oligonucleotide primer 17A-BR, 3'
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:
CCCGGATCCG AATTCTTACC CATCATCGGC AAACCAG 37
(2) INFORMATION FOR SEQ ID NO:72:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: oligonucleotide primer 1A-NF, 5'
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:
CGGCCATGGA ATTCGAACTC GTTGAGAGC 29
(2) INFORMATION FOR SEQ ID NO:73:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: oligonucleotide primer 1A-BR, 3'
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:
CCCGGATCCG AATTCTTAAA CCGTGTTTTC GGATCC 36
(2) INFORMATION FOR SEQ ID NO:74:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 109 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: GB Clone 487-13-5
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:
GAATTCCCTA GGCGTGGGGC ATCGCTATCA AAAAGTGATG CGTCAGGTTC ATCGGGGCAG 60
AATCGGAAGC CATATGCCTC CGATTTTACT GGCCGTCGAC AACGAATTC 109
(2) INFORMATION FOR SEQ ID NO:75: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 126 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: GB Clone 487-14-11
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:
GAATTCCCTA GGCGTGGGGT CAGGGTCTCG CCCATCGGCA TCGTGACCGG CGCGGCGCCG 60
GGGCGCGTCA ATTCCACGCG TACCACGCGC ACCGGCTGGC CCAGGTAGGC GGCATCGAAC 120
GAATTC 126
(2) INFORMATION FOR SEQ ID NO:76:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 174 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: GB Clone 487-14-15
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:76: 80
134 GAA TTC CCT AGG CGT GGG GAA CAA CAG TAT GAA GAT GTT CTA.CAT GGG 48
GCC AAG TCG GTA TCT AAG ATT GTC ACC GAC TCT TCA GAT GAT GTC TTA 96
GCT AAG CAA AAG GTC TAT GAA GGA TCT AAG GGT GAT AAC CTA GTC ATG 144
ACC ATG GAC ATC GAT TTC CAG AAC GAA TTC 174
(2) INFORMATION FOR SEQ ID NO:77:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 99 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: GB Clone 487-15-7
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:
GAA TTC CCT AGG CGT GGG GAA GAG AAG ATG AGA AAT GAC AGA AAA GAA 48
AAT AAA CAA ATT AAA TTG AAG TAT CAA CAA AAA ATG GAA GAC AAC GAA 96
TTC 99
(2) INFORMATION FOR SEQ ID NO:78:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 163 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: GB Clone 487-19-7
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:
GAA TTC GTT ATA GGG AAA TCC AAA GAC AAC AGT GAA AAC ATA AAC AAG 48
AAC AGT AGA AGA AAT CTT TAG GGA AAT CCA AAG ACA ACA GTG AAA ACA 96
TAA ACA AGA ACA GTA GAA GAA AAT TTA GCC TAA GAT AGA GAA GAC ATG 144
GTC CAC GCC TAG GGA ATT C 163
(2) INFORMATION FOR SEQ ID NO:79:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 151 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: GB Clone 487-4-1
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:
GAA TTC CCT AGG CGT GGC GAT AAC ACG ATT TCT AAC AAT GCA GCC AAG 48
CAG GTG TTT GAA GGC TTG TGG GCG GGC GAG GGC GAG GTA GAC GCG ATT 96
ATC GAA GCC AAA GGC CTC AAG CAG GTG TCT GAT ACA GGC GCG ATT GAA 144
CGA ATT C 151 (2) INFORMATION FOR SEQ ID NO:80:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 165 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) , INDIVIDUAL ISOLATE: GB Clone 487-6-1
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:
GAATTCGTTG TTCGTCGTGT GACCGGTCGG TCAGAACCTC GGGTGGTTAC CCAGCAGCAG 60
CGCTCGCTCA TCGGCGACGT CCTTGCCCGC AGCCTTCTTG ACCGTGCCGA GAGTCCAGCA 120
GTCGATGTGG CGTGCGGTCA GTACCGCCCC ACGCCTAGGG AATTC 165
(2) INFORMATION FOR SEQ ID NO:81:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 125 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: GB Clone 487-6-10 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:
GAA TTC GTT CGG ACA GCT CGC CAA CCT CAA CCA GAT GAC GTC GCA GAG 48
CTC GGC CGG CGC CTC CGT CAT CAC CCT GCA GTT CAG CCT GGA TCT CGC 96
CCT CGA CAT CGC CCA CAC CTA GGG AAT TC 125
(2) INFORMATION FOR SEQ ID NO:82:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 229 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: GB Clone 487-6-2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:
GAA TTC CCT AGG CGT GGA GAA AGT TCT AGT GGT CTG GAT AAA AGA TCA 48
AAT CAG CCC CAA CAA GCC CTT AAG CCA AAA CCT AAT CCA GGG CAA GGC 96
TCT AGA AAG TTC TAG TGG TCA CCG AAT TCC CTA GGC GTG GGG ACA TGA 144
CAC ACC CGG CCA TGA GAT AAA TCA CAT TCA GTG CCA TAT TAA AAA AAA 192
TTA ATG CAA ACA AAA GTC CAT TAT CAA CAA CGA ATT C 229
(2) INFORMATION FOR SEQ ID NO:83:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 151 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: GB Clone 487-9-2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:83:
GAA TTC CCT AGG CGT GGC GAT AAC ACG ATT TCT AAC AAT GCA GCC AAG 48
CAG GTG TTT GAA GGC TTG TGG GCG GGC GAG GGC GAG GTA GAC GCG ATT 96
ATC GAA GCC AAA GGC CTC AAG CAG GTG TCT GAT ACA GGC GCG ATT GAA 144
CGA ATT C 151
(2) INFORMATION FOR SEQ ID NO:84:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 105 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: SCH Clone SC1-2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:84:
GAA TTC GCG GCC GCT CGA GGC TGC GGT GGA CAG AGA TCA GGC CAT TCC 48 AAG GCG AAC AAA GCT TCA TAC GCT TCT CCA AGT CAT CCC AAC CCG CGA 96
GAG GAA TTC 105
(2) INFORMATION FOR SEQ ID NO:85:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 123 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: SCH Clone SC5-2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:85:
GAA TTC CTT GAC GAA GAA GAG GAG GCG CTC TAC CTC AGC GCG CAA TTG 48
AAC TTG AGC GAG CGC GCC CGC GAA CTC GTT CAG CGG TTC GAA CTG GGC 96
GAA GGC GTC ACG AGC GGC CGC GAA TTC 123
(2) INFORMATION FOR SEQ ID NO:86:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 229 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: SCH Clone SUl-1
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:86:
GAA TTC GCG GCC GCT CGG CGG GAT GGC ACA AGG CTC AAC TCT TCT TCG 48
ACG CAA ACA TTC TAA CAG AAC GGA TCG GGA ACA AAC AAA CTT CGC CGC 96
GCG CTT CAT TGC GCG TCC GGT GCT GAA CGC GCC CGC GTC GAA CTC GAG 144
CCT GTG ATG ACG GGC GAT TCA CCC ATG CTT CGC GTT GCG GAG AAT GCA 192
AGC CGG TCC CTC TCG CGG CTC GAG CGG CCG CGA ATT C 229
(2) INFORMATION FOR SEQ ID NO:87:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 231 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: SCH Clone SU2-10
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:87:
GAA TTC GCG GCC GCT CGA CCC ATC GCG CTC CGG CGT CGG GAT CAG ACG 48
GCG TAT GTT GTA GAA GAC GAT CTC GCG TGT TGC GAC GAA GAA CCC ATC 96
GCG CTC CGG CGT CGG GAT CAG ACG GCG TAT GTT GTA GCC TTC CAT GCC 144
GAA AAG CTT CTG CGG ACG CTG GCC CGG AAT GAA CGA GTA GAC GAA CCC 192
GCT GAT GTA GTA AAC GAT GTC GCG AGC GGC CGC GAA TTC 231 (2) INFORMATION FOR SEQ ID NO:88:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 358 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: SCH Clone SU2-4
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:88:
GAA TTC GCG GCC GCT CGG TCC GAA GTT GAC ACA AGC ATG AAG TCA AGT 48
GGT CCA AGG CAA CTG ACT TCT GAA CGT TCA GTT AGA ACC CCA AGG AGA 96
ACT GCA AAT GAA CAT TTG TCG TCG CAT ATT GGC AGC TTC CGT TCT GTC 144
TGC GTC TCT CAC CCT TCC TGT CTT AGC GGG CGA GAT GGA CAC CGG TTA 192
TAC ACC ACC ACC GCC AAC CAC CAA CGC TGC GGG CGA GAT GGA CAC TGG 240
CTA TGC CGG GCA GAT GGA CAC GGG CTA CGC GGG AGA GAT GGG CAC TGG 288
AGC AGC TCA AAC ATC ATC TGC TAG CTC GAT CAC AGA GAT TTT GCT TCT 336
TAT CAC GAG CGG CCG CGA ATT C 358
(2) INFORMATION FOR SEQ ID NO:89:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 300 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: SCH Clone SU2-5
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:89:
GAA TTC GCG GCC GCT CGG CGG GTC AAT GAG AGC GCA GGC AAC CGG CAC 48
CCG GAA GTG ATC CCA ACT GGC AAT CAG CAA CAC CAT CTC AAA GCC GAA 96
CGA GAA GCG GGC GTA CTC GTT CAT GCG GCA TTT GCG CCC GAG CGG GTG 144
TTT CTC ACC ACG TTT CCC CTT AAG TGT CGC GTC ACC GAT CAG ATG CAG 192
GGT GGC ATC CTG GGG CGG AGG CAA AAG GCG CAA GGT GTC GGC GCT CAA 240
CTC TTT AAC CAG TGC TCG TTC ATC CCA CCA GCC GCT TCT GAC CCG AGC 288
GGC CGC GAA TTC 300
(2) INFORMATION FOR SEQ ID NO:90:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 342 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: SCH Clone SU2-7
(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:90: GAA TTC GCG GCC GCT CGG GCC ATT GAA CAG GAT TAT CCT GCT CCG AAA 48
CAG AGG GAA GAG CAT ACC CCT GAT AAA CAA CCT CTC TCT TCC AAC ATA 96
CAA ACT GAT TCC GTG GAT CCT GAA CAG AGC AAT CCG AAC CGT TTC TGT 144
CTT CTG TGC AAA GAA TTT TAT GGT TTC AGG TAC ATC CTT GAT AAC AAC 192
GGA AAC ACC GTT GTT CGT CGC TGC TCA CAC GTC CCC GAA GTA GAG AAG 240
AGG TTC AAG CCC TCG GGA AAC CTC TCC CAG TCA TAG ACC CCA CGC CTA 288
AAG GCG GGG GCT TGC GGG TAT GAC CCG GCA AGC CTG TCG AGC GGC CGC 336
GAA TTC 342
(2) INFORMATION FOR SEQ ID NO:91:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 122 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: SCH Clone SU2-9
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:91:
GAA TTC GCG GCC AGT CGA GCC ACG GAA ATT CAC TTG CAG CAC GGC GTA 48
GCC ACG GTT AGC GAG CCA CTG AAC CAT CGG GTT ATA GCC CCA CGT GTC 96
GCG CGA CCA CGA GCG GCC GCG AAT TC 122
(2) INFORMATION FOR SEQ ID NO:92: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 305 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(Vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: SCH Clone SU4-3
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:92:
GAA TTC GCG GCC GCT CGG GGG CAC GCA CAT GCT GAA GCG AGG GTA ACA 48
ACG ACA ACG TCG AAT CAG ACG GGC AGC AGG AGT GCT GAT ACG CCA AAG 96
AGC GCA GGA AGT TGG AAG TTT GAC CCG AAG GTG GAT TTG GAT TGG CGC 144
GGT ACT GGC AAA ACT GTT AGG GAA GCA GTG GAT GAG GCT TTC AAA CGC 192
ACT GGT GTT CCT AAA GAG GAT TTC GAG GTA ACG AAA TGG GCC GTT GAT 240
AAA AAT GGC AAA AGT TTT CCT GTC GAA TGG AGA GCA AAA GGT GGA GCA 288
CGA GCG GCC GCG AAT TC 305
(2) INFORMATION FOR SEQ ID NO:93:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 240 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: SCH Clone SU4-4
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:93:
GAA TTC GCG GCC GCT CGG CGG GCG CTA GAG GCC GGC GCT TTG TTG CAC 48
GAT ATC GGC AAA CTC GCC GTG CCC GAA TAC ATT CTG AAC AAG CCG GGC 96
AAA TTG ACC GCG GCC GAG TTT GAG AAG ATG AAA GTC CAC ACG GTG GTC 144
GGT GCC GAC ATC GTC CGG CGC GTT GGA TTT CCT TAC CCG GTG GAA GAC 192
ATC GTC CGC TAC CAC CAC GAG AAA TGG GAC GCG AGC GGC CGC GAA TTC 240
(2) INFORMATION FOR SEQ ID NO:94:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 318 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: SCH Clone SU5-4
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:94:
GAA TTC GCG GCC GCT CGG CGG GCA CTG CTA CAA TTT GCA CAG AGT CAA 48
TAT CCT GAT GGT AAA GTT TCT GTG GTG ATT GGT GCA CCA GGC AAT AAA 96
GGT GTG TCA CGC CGT GCT GAC TTT GGT CGG GTG GTG TCA GAG TTT GCG 144
GAT ACG GTC TTT TTG ACG GCT GAT GAT CCA CAA TTT GAA TCG CCA ATG 192 GCC ATT GCC AAA GAA ATT GCG GCC CAC ATT ACC AAT CCT GAT GTG ACG 240
GTA CAT TTT GAA ATG GAT CGT ATC CAA GCC ATT CAA CAG GCG ATT GCC 288
CAG GCT AAC CCG CCG AGC GGC CGC GAA TTC 318
(2) INFORMATION FOR SEQ ID NO:95:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 189 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: SCH Clone SU5-6
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:95:
GAA TTC GCG GCC GCT CGA CGG CAC AAA CTC TCC GCC CGC TAC CTG GAT 48
GAT GAT TCG ATC ACT ACG CCG AGC GCG ATG AAC TCG CCT TTC TTC ACC 96
ACC GAG TTC AAC GGC ATC TCG CGC AAC CTT CTG TTC ACC TAC ACG TGG 144
GTA GTC AAT CCG ACC ATC ACC AAC GAG CTG CGC GGC CGC GAA TTC 189
(2) INFORMATION FOR SEQ ID NO:96:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 210 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: SCH Clone SU6-2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:96:
GAA TTC GCG GCC GCT CGG GCG GCG AGT TCG AGC AAC GCG GCG AGG CGT 48
TTA AAT TCA GAA GTG TGC GAT GGG GTT TGG CGT AGG AAA TCG ATC CAG 96
AGC GGA GCG TTA AAG AGG AGG CCA TCG ATC CGT CCA AGT GCG GTT TGA 144
TCG ACG ATC AGA TCA ATA GGA TCA AAA CAA GTC CAA TCA GCG AGT TGT 192
GCG AGC GGC CGC GAA TTC 210
(2) INFORMATION FOR SEQ ID NO:97:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 234 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: SCH Clone SU6-4
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:97:
GAA TTC GCG GCC GCT CGG GGC GAA GCG GAT CTG TAC TAC AAG ACG GCC 48
GAC GGC GTG GTA CAT GTG GAA GAG GTC AAG AGC ACG TTT CGG GCA TTG 96
AAC CGT AAG TTG GAA AAG GTG AAG AAG GTA GCT GAA GGA GCC AGA GAC 144 GAT CGA GAG AAG CTG CAA AGA ATC ATG AAG GAT ACG CAG TTG GGG CGG 192
TAT GTG AAG TGG GAG CAA GAA GGG GCG AGC GGC CGC GAA TTC 234
(2) INFORMATION FOR SEQ ID NO:98:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 294 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: SCH Clone SU7-8
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:98:
GAA TTC GCG GCC GCT CGC GGC CAG TTA CCC GAA GGG GAA CCG CTG CTC 48
GCG ACG GAT GAA GTT ACC ATC TAC GGC CCG CGT TCC GAT GAG ATC AAA 96
TCC GGC AAT GCG CGA ACG CTA ATC AGG CAA CAC ATC GCG CAA GCG CGC 144
GCC GGG CGC GAT TAC GTC GCG CTG CTC CTC TAT CTC GAA GAG GCA GCA 192
GAA CAC GAT GCT TTG GTT CGC GAC ATT CGC CGT CAT GTA CGC GAT CGC 240
CTG CGC GTG GCG ACC ACC GCT GGT TAC GGT CCG CGC TCG AGC GGC CGC 288
GAA TTC 294
(2) INFORMATION FOR SEQ ID NO:99:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 273 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: SCH Clone SU7-9
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:99:
GAA TTC GCG GCC GCT CGG GCG GAC CGA TGG CCC GAA CGA TGG TCC GGT 48
TTG CGA CCC ACG CAC GGG AAG AGC CTT TCC CGG CGG CAT CAT CCC GTT 96
GAG TCG CCA GAC AAC GGT CGG GCG GAA CTA CCT GAA CGC CTT TCC GCT 144
GCC GAC GCG CAA CGT CTT CAA CCC GAG CGA CTC GCT CGA AGC CCG CAA 192
CTA CTT CAC GCA GCG CGC TAA TCG CGA GAT CAT CAA CAA CTT CGG GCT 240
GCG CAT CGA CCA CCG TTT CAG CGA AAG GAA TTC 273
(2) INFORMATION FOR SEQ ID NO:100:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 198 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: SCH Clone SU8-3 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:100:
GAA TTC GCG GCC GCT CGA TGC GTG GTC TAT GAC GAT CAG CAC GGC CCG 48
GCG CTT GCA CGC CGA GAT GGT TCG GCT GTT GCA GCA GAG CGA AAG ATG 96
GTG AGC CTA GCG CGG GTA AAG TCC AAC TCT GCT GCT GCT GCG CGT TTG 144
AGT TGC AGC GCG GCG AGA GTT GGT GAG CTT GGT GGG GCG AGC GGC CGC 192
GAA TTC 198
(2) INFORMATION FOR SEQ ID NO:101:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 268 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: SCH Clone SU8-4
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:101:
GAA TTC GCG GCC GCT CGA AAA CTC AAC GCA TAC CAG ACT GAA CCG ACC 48
CAG CCG GCT AAG GCT TGA CCA GAT AAC CCA ATG TTA AAG AAT CCA GCT 96
GTT TGC GCC ACA GCA AAA CCA AGC GCT GTT AAA ATC AAT GGC GTC ATT 144
TGA GTC AAG ACA CCA CCG ATG TCT TGC ATT GAA CCA AAG GCT GAA CCC 192
AGT AAC GCC ACA TAA CCA GAA ATC GGA TTA TAC CCG AAA ACC AGC ATA 240
ATA ACG GCC CCC GAG CGG CCG CGA ATT C 268 (2) INFORMATION FOR SEQ ID NO:102:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 345 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: SCH Clone SU8-7
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:102:
GAA TTC GCG GCC GCT CGC CCA ACT AAC CCG CCG CCA CCA TCG CCA TCG 48
GCC GCA AAA TGT GGC ACC AAG AGA CGC TCC CCA TCC AGC AAA ACC ACC 96
CAG TCA GCT TTA AAG GTT GCC TCA CAA GCT AGC GCC CCT TTT TCA AAC 144
GAA GGC TCC GGC ATA AAA CTT GCT CCC GAA AAA AAC GGA GAA TTA CGG 192
GGG AAA AAC GCC CGT GCT GAC GGG CCG TGC TTT TTG CCT GGT AAT ATC 240
CAC ATA GTC TTG AAA AAA GTG GTC TTC CGA TGC CGC CGC AAA GCG CCG 288
CCG AAC TCG AAG CGG AGA TCA AGC GCG CCG AAG AAC TTC TCG AGC GGC 336
CGC GAA TTC 345
(2) INFORMATION FOR SEQ ID NO:103:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 258 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: Clone 2DR8
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:103:
GAATTCGCGG CCGCTCGATG TATCAAAGCT AATTCAAATG ACTATAGAAG CCAAAATGGC 60
GCCCTTTGGC TCTCACCAAA GGGCGCTTTC TTGTAGAAAG AGGAACCTAT GTCAACTTTT 120
TCCGGATTTT ATAAAAAATC ACGCCAAGAA CGCATTGATA TTCTCCAACA GAATCGTTCC 180
CTATCCGAAG ATAGCTTGGA CATTCTATAC AAAGACGAAA ACCTTCCAGA AGCAATTGCA 240
GCGAGCGGCC GCGAATTC 258
(2) INFORMATION FOR SEQ ID NO:104:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 430 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: Clone 470-20-1 extension sequence
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2..430 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:104:
A GCA ACA TCA GCC ACC GTC AAC CCC AAT GAG AAA AAG CGC GTG ACG 46
Ala Thr Ser Ala Thr Val Asn Pro Asn Glu Lys Lys Arg Val Thr 1 5 10 15
CTC TTT TCA ACG CAG CAC GAC ATC TTG ACG GTA AGC TTC CTG GTC GCG 94 Leu Phe Ser Thr Gin His Asp lie Leu Thr Val Ser Phe Leu Val Ala 20 25 30
TCG CTC TGT GGA AAT AAG GCT TTT AAT ACG GAA AGA GCC ACG TTG AAG 142 Ser Leu Cys Gly Asn Lys Ala Phe Asn Thr Glu Arg Ala Thr Leu Lys 35 40 45
ACA CTT TCC TCC CCT TCG GCT GTC TCG GAC TCT TGG ATG ACC TCG AAT 190 Thr Leu Ser Ser Pro Ser Ala Val Ser Asp Ser Trp Met Thr Ser Asn 50 55 60
GAG TCA GAG GAC GGG GTA TCC TCC TGC GAG GAG GAC ACC GAC GGG GTC 238 Glu Ser Glu Asp Gly Val Ser Ser Cys Glu Glu Asp Thr Asp Gly Val 65 70 75
TTC TCA TCT GAG CTG CTC TCA GTA ACC GAG ATA AGT GCT GGC GAT GGA 286 Phe Ser Ser Glu Leu Leu Ser Val Thr Glu lie Ser Ala Gly Asp Gly 80 85 90 95
GTA CGG GGG ATG TCT TCT CCC CAT ACA GGC ATC TCT CGG CTA CTA CCA 334 Val Arg Gly Met Ser Ser Pro His Thr Gly lie Ser Arg Leu Leu Pro 100 105 110
CAA AGA GAG GGT GTA CTG CAG TCC TCC ATG ATG ACA TCA ATG TGC GGT 382 Gin Arg Glu Gly Val Leu Gin Ser Ser Met Met Thr Ser Met Cys Gly 115 120 125
TCA AGA ATC CTC GCA GCA TTC TCG ATC GCT TGG AGA GCA GCA GCC GCC 430 Ser Arg lie Leu Ala Ala Phe Ser lie Ala Trp Arg Ala Ala Ala Ala 130 135 140
(2) INFORMATION FOR SEQ ID NO:105:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 143 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:105:
Ala Thr Ser Ala Thr Val Asn Pro Asn Glu Lys Lys Arg Val Thr Leu 1 5 10 15
Phe Ser Thr Gin His Asp lie Leu Thr Val Ser Phe Leu Val Ala Ser 20 25 30
Leu Cys Gly Asn Lys Ala Phe Asn Thr Glu Arg Ala Thr Leu Lys Thr 35 40 45
Leu Ser Ser Pro Ser Ala Val Ser Asp Ser Trp Met Thr Ser Asn Glu 50 55 60
Ser Glu Asp Gly Val Ser Ser Cys Glu Glu Asp Thr Asp Gly Val Phe 65 70 75 80
Ser Ser Glu Leu Leu Ser Val Thr Glu lie Ser Ala Gly Asp Gly Val 85 90 95
Arg Gly Met Ser Ser Pro His Thr Gly lie Ser Arg Leu Leu Pro Gin 100 105 110
Arg Glu Gly Val Leu Gin Ser Ser Met Met Thr Ser Met Cys Gly Ser 115 120 125
Arg lie Leu Ala Ala Phe Ser lie Ala Trp Arg Ala Ala Ala Ala 130 135 140
(2) INFORMATION FOR SEQ ID NO:106:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 237 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: PNF2161 Clone 470-20-1
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:106:
GAATTCGCGG CCGCTCGGGC TGTCTCGGAC TCTTGGATGA CCTCGAATGA GTCAGAGGAC 60
GGGGTATCCT CCTGCGAGGA GGACACCGAC GGGGTCTTCT CATCTGAGCT GCTCTCAGTA 120
ACCGAGATAA GTGCTGGCGA TGGAGTACGG GGGATGTCTT CTCCCCATAC AGGCATCTCT 180
CGGCTACTAC CACAAAGAGA GGGTGTACTG CAGTCCTCCA CGAGCGGCCG CGAATTC 237

Claims

IT IS CLAIMED:
1. A method of obtaining immunogenic polypeptides associated with non-A, non-B, non-C, non-D, non-E hepatitis agent (N-(ABCDE)) infection, comprising preparing phage from a library selected from the group consisting of MY 620 DNA source, MY 620 cDNA source, MY 670 DNA source, and MY 670 cDNA source, plating the phage to form plaques, and screening the phage plaques for production of poly- peptides immunoreactive with N-(ABCDE) serum.
2. The method of claim 1, wherein the N-(ABCDE) serum is selected from the group consisting of human serum, mystax monkey serum or cynomolgus monkey serum.
3. A method of obtaining non-A, non-B, non-C, non-D, non-E hepatitis agent (N-(ABCDE) ) coding sequences associated with a region of a N-(ABCDE) genome, comprising (a) preparing phage having insert sequences from a library selected from the group consisting of MY 620 DNA source, MY 620 cDNA source, MY 670 DNA source, and MY 670 cDNA source, plating the phage to form plaques, screening the phage plaques for production of polypeptides immunoreactive with N-(ABCDE) serum, isolating the phage that produce polypeptides immunoreactive with N-(ABCDE) serum and preparing the insert sequence for use as a hybridization probe, (b) preparing phage from a library selected from the group consisting of MY 620 DNA source, MY 620 cDNA source, MY 670 DNA source, and MY 670 cDNA source, plating the phage to form plaques, screening the phage plaques for hybridization to said hybridization probe.
4. A phage library selected from the group consisting of: MY 620 DNA source, MY 620 cDNA source, MY 670 DNA source, and MY 670 cDNA source.
PCT/US1995/005980 1994-05-20 1995-05-17 Non-a/non-b/non-c/non-d/non-e hepatitis agents and molecular cloning thereof WO1995032290A2 (en)

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US08/246,986 1994-05-20

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5709997A (en) * 1995-08-14 1998-01-20 Abbott Laboratories Nucleic acid detection of hepatitis GB virus
US5807670A (en) * 1995-08-14 1998-09-15 Abbott Laboratories Detection of hepatitis GB virus genotypes
US5843450A (en) * 1994-02-14 1998-12-01 Abbott Laboratories Hepatitis GB Virus synthetic peptides and uses thereof
US5955318A (en) * 1995-08-14 1999-09-21 Abbott Laboratories Reagents and methods useful for controlling the translation of hepatitis GBV proteins
US5981172A (en) * 1994-02-14 1999-11-09 Abbott Laboratories Non-A, non-B, non-C, non-D, non-E Hepatitis reagents and methods for their use
US6051374A (en) * 1994-02-14 2000-04-18 Abbott Laboratories Non-A, non-B, non-C, non-D, non-E hepatitis reagents and methods for their use
US6156495A (en) * 1994-02-14 2000-12-05 Abbott Laboratories Hepatitis GB virus recombinant proteins and uses thereof
US6451578B1 (en) 1994-02-14 2002-09-17 Abbott Laboratories Non-A, non-B, non-C, non-D, non-E hepatitis reagents and methods for their use
US6558898B1 (en) 1994-02-14 2003-05-06 Abbott Laboratories Non-A, non-B, non-C, non-D, non-E hepatitis reagents and methods for their use
US6586568B1 (en) 1994-02-14 2003-07-01 Abbott Laboratories Non-A, non-B, non-C, non-D, non-E hepatitis reagents and methods for their use
US6720166B2 (en) 1994-02-14 2004-04-13 Abbott Laboratories Non-a, non-b, non-c, non-c, non-d, non-e hepatitis reagents and methods for their use

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995021922A2 (en) * 1994-02-14 1995-08-17 Abbott Laboratories Non-a, non-b, non-c, non-d, non-e hepatitis reagents and methods for their use

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
WO1995021922A2 (en) * 1994-02-14 1995-08-17 Abbott Laboratories Non-a, non-b, non-c, non-d, non-e hepatitis reagents and methods for their use

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HEPATOLOGY, vol. 9, no. 2, February 1989 pages 186-192, P.KARAYIANNIS ET AL. 'Studies of GB hepatitis agent in tamarins' *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, vol. 92, no. 8, 11 April 1995 WASHINGTON US, pages 3401-3405, J.N.SIMONS ET AL. 'Identification of two flavivirus-like genomes in the GB hepatitis agent' *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5843450A (en) * 1994-02-14 1998-12-01 Abbott Laboratories Hepatitis GB Virus synthetic peptides and uses thereof
US5981172A (en) * 1994-02-14 1999-11-09 Abbott Laboratories Non-A, non-B, non-C, non-D, non-E Hepatitis reagents and methods for their use
US6051374A (en) * 1994-02-14 2000-04-18 Abbott Laboratories Non-A, non-B, non-C, non-D, non-E hepatitis reagents and methods for their use
US6156495A (en) * 1994-02-14 2000-12-05 Abbott Laboratories Hepatitis GB virus recombinant proteins and uses thereof
US6451578B1 (en) 1994-02-14 2002-09-17 Abbott Laboratories Non-A, non-B, non-C, non-D, non-E hepatitis reagents and methods for their use
US6558898B1 (en) 1994-02-14 2003-05-06 Abbott Laboratories Non-A, non-B, non-C, non-D, non-E hepatitis reagents and methods for their use
US6586568B1 (en) 1994-02-14 2003-07-01 Abbott Laboratories Non-A, non-B, non-C, non-D, non-E hepatitis reagents and methods for their use
US6720166B2 (en) 1994-02-14 2004-04-13 Abbott Laboratories Non-a, non-b, non-c, non-c, non-d, non-e hepatitis reagents and methods for their use
US5709997A (en) * 1995-08-14 1998-01-20 Abbott Laboratories Nucleic acid detection of hepatitis GB virus
US5807670A (en) * 1995-08-14 1998-09-15 Abbott Laboratories Detection of hepatitis GB virus genotypes
US5955318A (en) * 1995-08-14 1999-09-21 Abbott Laboratories Reagents and methods useful for controlling the translation of hepatitis GBV proteins

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AU2549195A (en) 1995-12-18

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