U.S. patent application number 10/621675 was filed with the patent office on 2005-03-03 for process for the determination of peptides corresponding to immunologically important epitopes and their use in a process for determination of antibodies or biotinylated peptides corresponding to immunologically important epitopes, a process for preparing them and compositions containing them.
This patent application is currently assigned to N.V. INNOGENETICS S.A.. Invention is credited to De Leys, Robert.
Application Number | 20050049398 10/621675 |
Document ID | / |
Family ID | 29737197 |
Filed Date | 2005-03-03 |
United States Patent
Application |
20050049398 |
Kind Code |
A1 |
De Leys, Robert |
March 3, 2005 |
Process for the determination of peptides corresponding to
immunologically important epitopes and their use in a process for
determination of antibodies or biotinylated peptides corresponding
to immunologically important epitopes, a process for preparing them
and compositions containing them
Abstract
The technical problem underlying the present invention is to
provide peptides corresponding to immunologically important
epitopes on bacterial and viral proteins, as well as the use of
said peptides in diagnostic or immunogenic compositions. The
invention relates to a process for the in vitro determination of
antibodies, wherein the peptides used are biotinylated,
particularly in the form of complexes of streptavidin-biotinylated
peptides or of avidin-biotinylated peptides.
Inventors: |
De Leys, Robert;
(Grimbergen, BE) |
Correspondence
Address: |
NIXON & VANDERHYE, PC
1100 N GLEBE ROAD
8TH FLOOR
ARLINGTON
VA
22201-4714
US
|
Assignee: |
N.V. INNOGENETICS S.A.
|
Family ID: |
29737197 |
Appl. No.: |
10/621675 |
Filed: |
July 18, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10621675 |
Jul 18, 2003 |
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09576824 |
May 23, 2000 |
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6667387 |
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09576824 |
May 23, 2000 |
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08723425 |
Sep 30, 1996 |
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6165730 |
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08723425 |
Sep 30, 1996 |
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08146028 |
Nov 22, 1993 |
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5891640 |
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08146028 |
Nov 22, 1993 |
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PCT/EP93/00517 |
Mar 8, 1993 |
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Current U.S.
Class: |
530/350 |
Current CPC
Class: |
G01N 33/56983 20130101;
C07K 2319/00 20130101; G01N 2333/18 20130101; C12N 2740/16222
20130101; G01N 33/56988 20130101; G01N 33/6878 20130101; C12N
2740/16122 20130101; C12N 2740/16022 20130101; C12N 2770/24222
20130101; C07K 14/005 20130101; G01N 33/531 20130101; C07K 1/13
20130101 |
Class at
Publication: |
530/350 |
International
Class: |
C07K 014/705 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 6, 1992 |
EP |
92400598.6 |
Claims
1-22. (Canceled)
23. The peptide consisting of an amino acid sequence selected from
the group consisting of:
80 IPKPQRKTK, (SEQ ID NO 181) PKPQRKTKR, (SEQ ID NO 182) KPQRKTKRN,
(SEQ ID NO 183) PQRKTKRNT, (SEQ ID NO 184) QRKTKRNTN, (SEQ ID NO
185) RKTKRNTNR, (SEQ ID NO 186) KTKRNTNRR, (SEQ ID NO 187)
TKRNTNRRP, (SEQ ID NO 188) RRPQDVKFP, (SEQ ID NO 194) RPQDVKFPG,
(SEQ ID NO 195) PQDVKFPGG, (SEQ ID NO 196) QDVKFPGGG, (SEQ ID NO
197) DVKFPGGGQ, (SEQ ID NO 199) GGVYLLPRR, (SEQ ID NO 209)
GVYLLPRRG, (SEQ ID NO 210) VYLLPRRGP, (SEQ ID NO 211) YLLPRRGPR,
(SEQ ID NO 212) LLPRRGPRL, (SEQ ID NO 213) LPRRGPRLG, (SEQ ID NO
214) PRRGPRLGV, (SEQ ID NO 215) GPRLGVRAT, (SEQ ID NO 218)
PRLGVRATR, (SEQ ID NO 219) RLGVRATRK, (SEQ ID NO 220) ERSQPRGRR,
(SEQ ID NO 231) RSQPRGRRQ, (SEQ ID NO 232) SQPRGRRQP, (SEQ ID NO
233) RGRRQPIPK, (SEQ ID NO 236) GRRQPIPKV, (SEQ ID NO 237)
RRQPIPKVR, (SEQ ID NO 238) PIPKVRRPE, (SEQ ID NO 241) PEGRTWAQP,
(SEQ ID NO 248) EGRTWAQPG, (SEQ ID NO 249) GRTWAQPGY, (SEQ ID NO
250) RTWAQPGYP, (SEQ ID NO 251) TWAQPGYPW, (SEQ ID NO 252)
WAQPGYPWP, (SEQ ID NO 253) AQPGYPWPL, (SEQ ID NO 254) QPGYPWPLY,
(SEQ ID NO 255) LSGKPAIIP, (SEQ ID NO 258) GKPAIIPDR, (SEQ ID NO
260) PAIIPDREV, (SEQ ID NO 263) AIIPDREVL, (SEQ ID NO 264)
IIPDREVLY, (SEQ ID NO 265) IPDREVLYR, (SEQ ID NO 266) PDREVLYRE,
(SEQ ID NO 267) DREVLYREF, (SEQ ID NO 268) CSQHLPYIE, (SEQ ID NO
281) SQHLPYIEQ, (SEQ ID NO 282) QHLPYIEQG, (SEQ ID NO 283)
HLPYIEQGM, (SEQ ID NO 284) LPYIEQGMM, (SEQ ID NO 285) PYIEQGMML,
(SEQ ID NO 286) YIEQGMMLA, (SEQ ID NO 287) IEQGMMLAE, (SEQ ID NO
288) MMLAEQFKQ, (SEQ ID NO 292) MLAEQFKQK, (SEQ ID NO 293)
LAEQFKQKA, (SEQ ID NO 294) AEQFKQKAL, (SEQ ID NO 295) EQFKQKALG,
(SEQ ID NO 296) QFKQKALGL, (SEQ ID NO 297) FKQKALGLL, (SEQ ID NO
298) KQKALGLLQ, (SEQ ID NO 299) QKALGLLQT, (SEQ ID NO 300)
KALGLLQTA, (SEQ ID NO 301) ALGLLQTAS, (SEQ ID NO 302) LGLLQTASR,
(SEQ ID NO 303) GLLQTASRQ, (SEQ ID NO 316) LLQTASRQA, (SEQ ID NO
317) SVPAEILRK, (SEQ ID NO 348) VPAEILRKS, (SEQ ID NO 349)
PAEILRKSR, (SEQ ID NO 350) AEILRKSRR, (SEQ ID NO 351) EILRKSRRF,
(SEQ ID NO 352) FAQALPVWA, (SEQ ID NO 360) AQALPVWAR, (SEQ ID NO
361) QALPVWARP, (SEQ ID NO 362) ALPVWARPD, (SEQ ID NO 363)
VWARPDYNP, (SEQ ID NO 366) WARPDYNPP, (SEQ ID NO 367) ARPDYNPPL,
(SEQ ID NO 368) RPDYNPPLV, (SEQ ID NO 369) PDYNPPLVE, (SEQ ID NO
370) PPLVETWKK, (SEQ ID NO 374) PLVETWKKP, (SEQ ID NO 375)
LVETWKKPD, (SEQ ID NO 376) VETWKKPDY, (SEQ ID NO 377) ETWKKPDYE,
(SEQ ID NO 378) TWKKPDYEP, (SEQ ID NO 379) WKKPDYEPP, (SEQ ID NO
380) KKPDYEPPV, (SEQ ID NO 381) KPDYEPPVV, (SEQ ID NO 382)
PDYEPPVVH, (SEQ ID NO 383) DYEPPVVHG, (SEQ ID NO 384) YEPPVVHGC,
(SEQ ID NO 385) PPVVHGCPL, (SEQ ID NO 387) PVVHGCPLP, (SEQ ID NO
388) VVHGCPLPP, (SEQ ID NO 389) VHGCPLPPP, (SEQ ID NO 390)
HGCPLPPPK, (SEQ ID NO 391) SPPVPPPRK, (SEQ ID NO 400) PQRKTK, (SEQ
ID NO 422) KTKRNTN, (SEQ ID NO 423) PQDVKFP, (SEQ ID NO 424)
YLLPRR, (SEQ ID NO 425) PRRGPRL, (SEQ ID NO 426) RLGVRAT, (SEQ ID
NO 427) SQPRGRR, (SEQ ID NO 428) RRQPIPK, (SEQ ID NO 429) RTWAQP,
(SEQ ID NO 430) QPGYPWPL, (SEQ ID NO 431) PDREVL, (SEQ ID NO 432)
HLPYIE, (SEQ ID NO 433) YIEQGMML, (SEQ ID NO 434) AEQFKQK, (SEQ ID
NO 435) KQKALG, (SEQ ID NO 436) LGLLQTA, (SEQ ID NO 437) PAEILRK,
(SEQ ID NO 438) EILRKSR, (SEQ ID NO 439) QALPVWA, (SEQ ID NO 440)
PDYNPP, (SEQ ID NO 441) LVETWKK, (SEQ ID NO 442) DYEPPV, (SEQ ID NO
443) and HGCPL, (SEQ ID NO 444)
wherein any of said amino acids are optionally biotynylated
N-terminally, C-terminally or internally, directly or through a
linker Y, said linker consisting of 1 to 10 chemical entities
chosen from glycine residues, beta-alanine, 4-aminobutyric acid,
5-aminovaleric acid or 6-aminohexanoic acid.
24. The peptide according to claim 23 which is coupled to
streptavidin or avidin, with said streptavidin or avidin optionally
coupled to a solid phase.
25. The peptide according to claim 23 wherein said peptide is
anchored to a solid support via covalent or non-covalent bonds.
26. The peptide according to claim 23, wherein said peptide is
coupled via its biotin group to streptavidin present on a nylon
membrane.
27. A method for detecting antibodies to HCV present in a
biological sample, comprising: (i) contacting the biological sample
to be analysed with a peptide according to any of claim 23 to 26,
and (ii) detecting an immune complex formed between said antibodies
to HCV and said peptide to determine the presence of antibodies to
HCV.
28. An immunological assay kit for detecting antibodies to HCV
comprising at least one peptide according to any of claims 23 to
26.
29. A Line Immunoassay kit for detecting antibodies to HCV
comprising at least one peptide according to any of claims 23 to
26.
30. A peptide consisting of an amino acid sequence as set forth in
any of SEQ ID NOs 477 to 599, and having the following chemical
structure:
81 (A)-IPKPQRKTK-(Z) (SEQ ID NO 477) (A)-PKPQRKTKR-(Z), (SEQ ID NO
478) (A)-KPQRKTKRN-(Z), (SEQ ID NO 479) (A)-PQRKTKRNT-(Z), (SEQ ID
NO 480) (A)-QRKTKRNTN-(Z), (SEQ ID NO 481) (A)-RKTKRNTNR-(Z), (SEQ
ID NO 482) (A)-KTKRNTNRR-(Z), (SEQ ID NO 483) (A)-TKRNTNRRP-(Z),
(SEQ ID NO 484) (A)-RRPQDVKFP-(Z), (SEQ ID NO 485)
(A)-RPQDVKFPG-(Z), (SEQ ID NO 486) (A)-PQDVKFPGG-(Z), (SEQ ID NO
487) (A)-QDVKFPGGG-(Z), (SEQ ID NO 488) (A)-DVKFPGGGQ-(Z), (SEQ ID
NO 489) (A)-GGVYLLPRR-(Z), (SEQ ID NO 490) (A)-GVYLLPRRG-(Z), (SEQ
ID NO 491) (A)-VYLLPRRGP-(Z), (SEQ ID NO 492) (A)-YLLPRRGPR-(Z),
(SEQ ID NO 493) (A)-LLPRRGPRL-(Z), (SEQ ID NO 494)
(A)-LPRRGPRLG-(Z), (SEQ ID NO 495) (A)-PRRGPRLGV-(Z), (SEQ ID NO
496) (A)-GPRLGVRAT-(Z), (SEQ ID NO 497) (A)-PRLGVRATR-(Z), (SEQ ID
NO 498) (A)-RLGVRATRK-(Z), (SEQ ID NO 499) (A)-ERSQPRGRR-(Z), (SEQ
ID NO 500) (A)-RSQPRGRRQ-(Z), (SEQ ID NO 501) (A)-SQPRGRRQP-(Z),
(SEQ ID NO 502) (A)-RGRRQPIPK-(Z), (SEQ ID NO 503)
(A)-GRRQPIPKV-(Z), (SEQ ID NO 504) (A)-RRQPIPKVR-(Z), (SEQ ID NO
505) (A)-PIPKVRRPE-(Z), (SEQ ID NO 506) (A)-PEGRTWAQP-(Z), (SEQ ID
NO 507) (A)-EGRTWAQPG-(Z), (SEQ ID NO 508) (A)-GRTWAQPGY-(Z), (SEQ
ID NO 509) (A)-RTWAQPGYP-(Z), (SEQ ID NO 510) (A)-TWAQPGYPW-(Z),
(SEQ ID NO 511) (A)-WAQPGYPWP-(Z), (SEQ ID NO 512)
(A)-AQPGYPWPL-(Z), (SEQ ID NO 513) (A)-QPGYPWPLY-(Z), (SEQ ID NO
514) (A)-LSGKPAIIP-(Z), (SEQ ID NO 515) (A)-GKPAIIPDR-(Z), (SEQ ID
NO 516) (A)-PAIIPDREV-(Z), (SEQ ID NO 517) (A)-AIIPDREVL-(Z), (SEQ
ID NO 518) (A)-IIPDREVLY-(Z), (SEQ ID NO 519) (A)-IPDREVLYR-(Z),
(SEQ ID NO 520) (A)-PDREVLYRE-(Z) (SEQ ID NO 521)
(A)-DREVLYREF-(Z), (SEQ ID NO 522) (A)-CSQHLPYIE-(Z), (SEQ ID NO
523) (A)-SQHLPYIEQ-(Z), (SEQ ID NO 524) (A)-QHLPYIEQG-(Z), (SEQ ID
NO 525) (A)-HLPYIEQGM-(Z), (SEQ ID NO 526) (A)-LPYIEQGMM-(Z), (SEQ
ID NO 527) (A)-PYIEQGMML-(Z), (SEQ ID NO 528) (A)-YIEQGMMLA-(Z),
(SEQ ID NO 529) (A)-IEQGMMLAE-(Z), (SEQ ID NO 530)
(A)-MMLAEQFKQ-(Z), (SEQ ID NO 531) (A)-MLAEQFKQK-(Z), (SEQ ID NO
532) (A)-LAEQFKQKA-(Z), (SEQ ID NO 533) (A)-AEQFKQKAL-(Z), (SEQ ID
NO 534) (A)-EQFKQKALG-(Z), (SEQ ID NO 535) (A)-QFKQKALGL-(Z), (SEQ
ID NO 536) (A)-FKQKALGLL-(Z), (SEQ ID NO 537) (A)-KQKALGLLQ-(Z),
(SEQ ID NO 538) (A)-QKALGLLQT-(Z), (SEQ ID NO 539)
(A)-KALGLLQTA-(Z), (SEQ ID NO 540) (A)-ALGLLQTAS-(Z), (SEQ ID NO
541) (A)-LGLLQTASR-(Z), (SEQ ID NO 542) (A)-GLLQTASRQ-(Z), (SEQ ID
NO 543) (A)-LLQTASRQA-(Z), (SEQ ID NO 544) (A)-SVPAEILRK-(Z), (SEQ
ID NO 545) (A)-VPAEILRKS-(Z), (SEQ ID NO 546) (A)-PAEILRKSR-(Z),
(SEQ ID NO 547) (A)-AEILRKSRR-(Z), (SEQ ID NO 548)
(A)-EILRKSRRF-(Z), (SEQ ID NO 549) (A)-FAQALPVWA-(Z), (SEQ ID NO
550) (A)-AQALPVWAR-(Z), (SEQ ID NO 551) (A)-QALPVWARP-(Z), (SEQ ID
NO 552) (A)-ALPVWARPD-(Z), (SEQ ID NO 553) (A)-VWARPDYNP-(Z), (SEQ
ID NO 554) (A)-WARPDYNPP-(Z), (SEQ ID NO 555) (A)-ARPDYNPPL-(Z),
(SEQ ID NO 556) (A)-RPDYNPPLV-(Z), (SEQ ID NO 557)
(A)-PDYNPPLVE-(Z), (SEQ ID NO 558) (A)-PPLVETWKK-(Z), (SEQ ID NO
559) (A)-PLVETWKKP-(Z), (SEQ ID NO 560) (A)-LVETWKKPD-(Z), (SEQ ID
NO 561) (A)-VETWKKPDY-(Z), (SEQ ID NO 562) (A)-ETWKKPDYE-(Z), (SEQ
ID NO 563) (A)-TWKKPDYEP-(Z), (SEQ ID NO 564) (A)-WKKPDYEPP-(Z),
(SEQ ID NO 565) (A)-KKPDYEPPV-(Z), (SEQ ID NO 566)
(A)-KPDYEPPVV-(Z), (SEQ ID NO 567) (A)-PDYEPPVVH-(Z), (SEQ ID NO
568) (A)-DYEPPVVHG-(Z), (SEQ ID NO 569) (A)-YEPPVVHGC-(Z), (SEQ ID
NO 570) (A)-PPVVHGCPL-(Z), (SEQ ID NO 571) (A)-PVVHGCPLP-(Z), (SEQ
ID NO 572) (A)-VVHGCPLPP-(Z), (SEQ ID NO 573) (A)-VHGCPLPPP-(Z),
(SEQ ID NO 574) (A)-HGCPLPPPK-(Z), (SEQ ID NO 575)
(A)-SPPVPPPRK-(Z), (SEQ ID NO 576) (A)-PQRKTK-(Z), (SEQ ID NO 577)
(A)-KTKRNTN-(Z), (SEQ ID NO 578) (A)-PQDVKFP-(Z), (SEQ ID NO 579)
(A)-YLLPRR-(Z), (SEQ ID NO 580) (A)-PRRGPRL-(Z), (SEQ ID NO 581)
(A)-RLGVRAT-(Z), (SEQ ID NO 582) (A)-SQPRGRR-(Z), (SEQ ID NO 583)
(A)-RRQPIPK-(Z), (SEQ ID NO 584) (A)-RTWAQP-(Z), (SEQ ID NO 585)
(A)-QPGYPWPL-(Z), (SEQ ID NO 586) (A)-PDREVL-(Z), (SEQ ID NO 587)
(A)-HLPYIE-(Z), (SEQ ID NO 588) (A)-YIEQGMML-(Z), (SEQ ID NO 589)
(A)-AEQFKQK-(Z), (SEQ ID NO 590) (A)-KQKALG-(Z), (SEQ ID NO 591)
(A)-LGLLQTA-(Z), (SEQ ID NO 592) (A)-PAEILRK-(Z), (SEQ ID NO 593)
(A)-EILRKSR-(Z), (SEQ ID NO 594) (A)-QALPVWA-(Z), (SEQ ID NO 595)
(A)-PDYNPP-(Z), (SEQ ID NO 596) (A)-LVETWKK-(Z), (SEQ ID NO 597)
(A)-DYEPPV-(Z), (SEQ ID NO 598) or (A)-HGCPL-(Z), (SEQ ID NO
599)
wherein A when present, represents an amino acid, amino group, or
chemically modified amino terminus of the peptide, and wherein Z
when present, represents an amino acid, OH-group, NH2-group, or a
linkage involving these two groups, and wherein any of said amino
acids are optionally biotynylated N-terminally, C-terminally or
internally, directly or through a linker Y, said linker consisting
of 1 to 10 chemical entities chosen from glycine residues,
beta-alanine, 4-aminobutyric acid, 5-aminovaleric acid or
6-aminohexanoic acid, or peptides derived from said chemical
structure which comprise a fragment which is immunologically
reactive with HCV antisera.
31. The peptide according to claim 30 which is coupled to
streptavidin or avidin, with said streptavidin or avidin optionally
coupled to a solid phase.
32. The peptide according to claim 30 wherein said peptide is
anchored to a solid support via covalent or non-covalent bonds.
33. The peptide according to claim 30, wherein said peptide is
coupled via its biotin group to streptavidin present on a nylon
membrane.
34. A method for detecting antibodies to HCV present in a
biological sample, comprising: (i) contacting the biological sample
to be analysed with a peptide according to any of claim 30 to 33,
and (ii) detecting an immune complex formed between said antibodies
to HCV and said peptide to determine the presence of antibodies to
HCV.
35. An immunological assay kit for detecting antibodies to HCV
comprising at least one peptide according to any of claims 30 to
33.
36. A Line Immunoassay kit for detecting antibodies to HCV
comprising at least one peptide according to any of claims 30 to
33.
Description
DETAILS
[0001] The technical problem underlying the present invention ell
is to provide peptides corresponding to immunologically important
epitopes on bacterial and viral proteins, as well as the use of
said peptides in diagnostic or immunogenic compositions.
[0002] Recent developments in genetic engineering as well as the
chemistry of solid phase peptide synthesis have led to the
increasingly wider use of synthetic peptides in biochemistry and
immunology. Protein sequences, which become available as a result
of molecular cloning techniques can be synthesized chemically in
large quantities for structural, functional, and immunological
studies. Peptides corresponding to immunologically important
epitopes found on viral and bacterial proteins have also proven to
be highly specific reagents, which can be used for antibody
detection and the diagnosis of infection.
[0003] Despite the many advantages synthetic peptides offer, there
are a number of disadvantages associated with their use. Because of
their relatively short size (generally less than 50 amino acids in
length), their structure may fluctuate between many different
conformations in the absence of the stabilizing influence of
intramolecular interactions present in the full-length protein.
[0004] Furthermore, the small size of these peptides means that
their chemical properties and solubilities will frequently be quite
different from those of the full-length protein and that the
contribution of individual amino acids in the peptide sequence
toward determining the overall chemical properties of the peptide
will be proportionally greater.
[0005] Many immunological assays require that the antigen used for
antibody detection be immobilized on a solid support. Most
enzyme-linked immunosorbent assays (ELISA) make use of polystyrene
as the solid phase.
[0006] Many proteins can be stably adsorbed to the solid phase and
present sequences, which are accessible for subsequent interactions
with antibodies. Because of their small size, direct adsorption of
peptides to the solid phase frequently gives rise to unsatisfactory
results for any of a number of reasons.
[0007] Firstly, the peptide may not possess the correct overall
charge or amino acid composition, which would enable the peptide to
bind to the solid phase. Secondly, the same amino acid residues,
which are required for binding to the solid phase, may also be
required for antibody recognition and therefore not available for
antibody binding. Thirdly, the peptide may become fixed in an
unfavourable conformation upon binding to the solid phase, which
renders it unrecognizable to antibody molecules. In many cases, it
is neither possible nor necessary to distinguish between these
possibilities. Binding to the solid phase can be increased and made
less sensitive to the specific chemical properties of a peptide by
first coupling the peptide to a large carrier molecule. Typically,
the carrier molecule is a protein.
[0008] While the amount of peptide bound to the solid phase, albeit
indirectly, can in some cases be increased by this method, this
approach suffers from the fact that the linkage between the peptide
and the carrier protein frequently involves the side chains of
internal trifunctional amino acids whose integrity may be
indispensable for recognition by antibodies. The binding avidity of
antisera for the internally modified peptide is frequently very
much reduced relative to the unmodified peptide or the native
protein.
[0009] The production of antisera to synthetic peptides also
requires in most cases that the peptide be coupled to a carrier.
Again, the coupling reaction between an internal trifunctional
amino acid of the peptide and the carrier is likely to alter the
immunogenic properties of the peptide.
[0010] There exist many methods for performing coupling reactions
and most of the procedures in current use are discussed in detail
in Van Regenmortel, M. H. V., Briand, J. P., Muller, S., and Plaud,
S.; Laboratory Techniques in Biochemistry, and Molecular Biology,
vol. 19, Synthetic Polypeptides as Antigens, Elsevier Press,
Amsterdam, N.Y., Oxford, 1988. In addition to these procedures,
unprotected peptides can also be biotinylated using commercially
available reagents such as N-hydroxysuccinimidobiotin or
biotinamidocaproate N-hydroxysuccinimide ester. Many of these
reagents are discussed in Billingsley, M. L., Pennypacker, K. R.,
Hoover, C. G., and Kincaid, R. L., Biotechniques (1987) 5(1):
22-31. Biotinylated peptides are capable of being bound by the
proteins streptavidin and avidin, two proteins, which exhibit
extraordinarily high affinity binding to biotin.
[0011] In certain instances, it is possible to selectively couple
biotin to an unprotected peptide or an unprotected peptide to a
carrier. This may be accomplished by synthesizing the peptide with
an additional trifunctional amino acid added to one of the ends,
which is capable of participating in the coupling reaction. This
approach will only be successful, however, as long as this amino
acid is not a critical residue in the immunogenic sequence of
interest and as long as the coupling agent chosen is sufficiently
selective. No single technique is applicable to all unprotected
peptides regardless of their amino acid composition.
[0012] The etiological agent responsible for non-A, non-B hepatitis
has been identified and termed hepatitis C virus (HCV). Patent
application EP-A-0318216 discloses sequences corresponding to
approximately 80% of the viral genome. The availability of these
sequences rapidly led to the elucidation of the remainder of the
coding sequences, particularly, those located in the 51 end of the
genome (Okamoto; J. Exp. Med. 60, 167-177,1990). The HCV genome is
a linear, positive-stranded RNA molecule with a length of
approximately 9400 nucleotides. With the exception of rather short
untranslated regions at the termini, the genome consists of one
large, uninterrupted, open reading frame encoding a polyprotein of
approximately 3000 amino acids. This polyprotein has been shown to
be cleaved cotranslationally into individual viral structural and
nonstructural (NS) regions. The structural protein region is
further divided into capsid (Core) and envelope (E1 and E2
proteins. The NS regions are divided into NS-1 to NS-5 regions.
[0013] A number of independent patent applications have employed a
variety of strategies to determine the locations of diagnostically
important amino acid sequences and many of these studies have led
to the identification of similar regions of the HCV
polyprotein.
[0014] The NS4 region has mainly been studied in EP-A-0 318 216,
EP-A-0 442 394,U.S. Pat. No. 5,106,726, EP-A-0 489 986, EP-A-0 484
787, and EP-A-0 445 801. Unfortunately only 70% of HCV-infected
individuals produce antibodies to NS4, neither the synthetic nor
recombinant proteins containing sequences from this region are
adequate for identifying all infected serum samples. The
nucleocapsid or Core region has been studied in patent applications
EP-A-0 442 394, US-5,106,726, EP-A-0 489 986, EP-A-0 445 801,
EP-A-0 451 891 and EP-A-0 479 376. It was found that these peptides
often used as mixtures were more frequently recognized by
antibodies (85-90%) in sera from chronically infected individuals
than were the peptides derived from NS4. The NS5 region was studied
in patent applications EP-A-0 489 986 and EP-A-0 468 527. Depending
on the serum panel used, more than 60% of NANB hepatitis can be
shown to contain antibodies directed against these peptides. The
NS3 region was also studied in patent application EP-A-0 468527.
All available evidence suggests that the most dominant epitope of
NS3 are discontinuous in nature and cannot be adequately
represented by synthetic peptides. The E1 region, which is
potentially interesting as a region from the outer surface of the
virus particles (possible immunogenic epitopes), was studied in
both patent applications EP-A: 0468 527 and EP-A-0507 615. The
E2/NSI region was studied for the same reason as E1. Comparisons of
this region from different HCV variants elucidated that this
protein contains variable region which are reminiscent of the HIV
V3 loop region of gp120 envelope protein. Four peptides were found
in EP-A 0468 527 which were shown to contain relatively
infrequently recognized epitopes. Finally, the NS2 region of HCV
was analyzed in EP-A-0 486 527. However, the diagnostic value of
this region is not clear yet. Virtually all patent applications
concerning diagnostically useful synthetic peptides for antibody
detection describe preferred combinations of peptides. Most of
these include peptides from the HCV core protein and NS4. In some
cases, peptides from NS5 (EP-A-0 489 968 and EP-A-0 468 527), and
E1 and E2/NS1 are included (EP-A-0 507 615 and EO-A-0 468 527).
[0015] Different patent applications have addressed the problem of
finding diagnostically useful epitopes of human immunodeficiency
virus (HIV). An important immunodominant region containing cyclic
HIV-1 and HIV-2 peptides was found in patent application EP-A-0 326
490. In EP-A-0 379 949, this region was asserted to be even more
reactive with HIV-specific antibodies in case a biotin molecule was
coupled to these cyclic HIV peptides. SU-A-161 22 64 also describes
the use of a biotinylated peptide in a solid phase immunoassay for
the detection of HIV antibodies.
[0016] Other applications have looked for useful HIV epitopes in
the hypervariable, V3 loop region of gp120 (such as EP-A-0 448 095
and EP-A-0 438 332). U.S. Pat. No. 4,833,071 provides peptide
compositions for detection of HTLV I antibodies.
[0017] Deciding whether or not an epitope is diagnostically useful
is not always straightforward and depends to an extent on the
specific configuration of the test into which it is incorporated.
It should be ideally an immunodominant epitope, which is recognized
by a large percentage of true positive sera or should be able to
complement other antigens in the test to increase the detection
rate.
[0018] Epitopes which are not frequently recognized may or may not
be diagnostically useful depending on the contribution they make
towards increasing the detection rate of antibodies in true
positive sera and the extent to which incorporation of these
epitopes has an adverse effect on the sensitivity of the test due
to dilution of other stronger epitopes.
[0019] Peptides can thus be used to identify regions of proteins,
which are specifically recognized by antibodies produced as a
result of infection or immunization. In general, there are two
strategies which can be followed. One of these strategies has been
described by Geysen, H. M., Meloen, R. H. and Barteling, S. J.;
Proc. Natl. Acad. Sci. USA (1984) 81:3998-4002. This approach
involves the synthesis of a large series of short, overlapping
peptides on polyethylene rods derivatized with a noncleavable
linker such that the entire length of the protein or protein
fragment of interest is represented. The rods are incubated with
antisera and antibody binding is detected using an
anti-immunoglobulin: enzyme conjugate. A positive reaction
immediately identifies the location and sequence of epitopes
present in the protein sequence. This technique has the advantage
that all peptides are uniformly linked to the solid support through
their carboxy-terminus. While this method allows for very accurate
mapping of linear epitopes, the length of the peptides, which can
be reliably synthesized on the rods, is limited. This may sometimes
present problems if the length of the epitope exceeds the length of
the peptides synthesized.
[0020] A second approach to epitope mapping involves the synthesis
of larger peptides, generally between fifteen and thirty amino
acids in length, along the sequence of the protein to be analyzed.
Consecutive peptides may be contiguous but are preferably
overlapping. Following cleavage, the evaluation of antibody binding
to the individual peptides is assessed and the approximate
positions of the epitopes can be identified. An example of this
approach is given in Neurath, A. R., Strick, N., and Lee, E. S. Y.;
J. Gen. Virol. (1990) 71:85-95. This approach has the advantage
that longer peptides can be synthesized which presumably more
closely, resemble the homologous sequence in the native protein and
which offer better targets for antibody binding. The disadvantage
of this approach is that each peptide is chemically unique and that
the conditions under which each peptide can be optimally coated
onto a solid phase for immunological evaluation may vary widely in
terms of such factors as pH, ionic strength, and buffer
composition. The quantity of peptide which can be adsorbed onto the
solid phase is also an uncontrolled factor which is unique for each
peptide.
[0021] The main purpose of the present invention is to provide
modified peptides corresponding to immunologically useful epitopes
with said modified peptides having superior immunological
properties over non-modified versions of these peptides.
[0022] Another aim of the present invention is to provide modified
peptides corresponding to immunologically useful epitopes, which
could not be identified through classical epitope mapping
techniques.
[0023] Another aim of the present invention is to provide a process
for the in vitro determination of antibodies using said peptides,
with said process being easy to perform and amenable to
standardization.
[0024] Another aim of the invention is to provide a process for the
determination of peptides corresponding to immunologically
important epitopes on bacterial and viral proteins.
[0025] Another aim of the invention is to provide a method for
preparing protein sequences used in any of said methods.
[0026] Another aim of the invention is to provide a method for
preparing protein sequence, which can be used in a process for the
determination of their epitopes or in an in vitro method for the
determination of antibodies.
[0027] Another aim of the invention is to provide intermediary
compounds useful for the preparation of peptides used in the
above-mentioned methods.
[0028] Another aim of the present invention is also to provide
compositions containing peptides determined to correspond to
immunologically important epitopes on proteins for diagnostic
purposes.
[0029] Another aim of the present invention is also to provide
compositions containing peptides determined to correspond to
immunologically important epitopes on proteins for vaccine
purposes.
[0030] According to the present invention, a series of biotinylated
peptides representing immunologically important regions of viral
proteins have been identified and prepared by solid phase peptide
synthesis. These peptides have been identified to be very useful
for (i) the detection of antibodies to HCV, and/or HIV, and/or
HTLV-I or II. In some preferred arrangements, these peptides were
also found or are at least expected, to be useful in stimulating
the production of antibodies to HCV, and/or HIV, and/or HTLV-I or
II in healthy animals such as BALB/C mice, and in a vaccine
composition to prevent HCV and/or HIV, and/or HTLV-I or II
infection.
[0031] As demonstrated in the Examples section of the present
invention, the use of biotinylated peptides also allows the
determination of immunologically important epitopes within a
previously determined protein sequence. The determination of
immunologically important epitopes using non-biotinylated peptides,
which are covalently coupled to the solid phase, often fails to
localize these epitopes.
[0032] Especially in case of localization of structural epitopes,
the use of biotinylated peptides seems to be quite successful.
[0033] (1) According to the present invention, a peptide
composition useful for the detection of antibodies to HCV, and/or
HIV, and/or HTLV-I or II comprise peptides corresponding to
immunologically important epitopes being of the structure:
(A)-(B)-(X)-(Y)-[amino acids].sub.n-Y-(X)-Z
[0034] where
[0035] [amino acids].sub.n is meant to designate the length of the
peptide chain where n is the number of residues, being an integer
from about 4 to about 50, preferably less than about 35, more
preferably less than about 30, and advantageously from about 4 to
about 25;
[0036] B represents biotin;
[0037] X represents a biotinylated compound, which is incorporated
during the synthetic process;
[0038] Y represents a covalent bond or one or more chemical
entities which singly or together form a linker arm separating the
amino acids of the peptide proper from the biotinyl moiety B or X,
the function of which is to minimize steric hindrance which may
interfere with the binding of the biotinyl moiety B or X to avidin
or streptavidin wherein Y is not a covalent bond, it is
advantageously at least one chemical entity and may consist of as
many as 30 chemical entities but will consist most frequently of 1
to 10 chemical entities, which may be identical or different, more
preferably glycine residues, .beta.-alanine, 4-aminobutyric acid,
5-aminovaleric acid, or 6-aminohexanoic acid;
[0039] B and X being enclosed in parentheses to indicate that the
presence of biotin or a biotinylated compound in these positions is
optional, the only stipulation being that B or X be present in at
least one of the positions shown;
[0040] A, when present, as indicated by parentheses, represents
(an) amino acid(s), an amino group, or a chemical modification of
the amino terminus of the peptide chain;
[0041] Z represents (an) amino acid(s), an OH-group, an NH2-group,
or a linkage involving either of these two chemical groups wherein
the amino acids are selectively chosen to be immunodominant
epitopes which are recognized by a large percentage of true
positive sera or are able to complement other antigens in the test
to increase the detection rate and B interacts with the selected
amino acids to produce a compound with greater diagnostic
sensitivity.
[0042] The peptide composition comprises at least one and
preferably a combination of two, three, four or more biotinylated
peptides chosen from the following sequences:
[0043] 1. Human Immunodeficiency Virus Type I Envelope
Peptides:
1 a. gp41 1. gp41, isolate HTLV-IIIB
(A)-(B)-(X)-Y-Ile-Trp-Gly-Cys-Ser-Gly-Lys-Ile-Cys-Y (X)-Z (SEQ ID
NO:1) 2. (A)-(B)-X)-Y-Ile-Trp-Gly-Cys-Ser-Gly-Lys-Leu-Il- e-Cys-
(SEQ ID NO:2) Thr-Thr-Ala-Val-Pro-Trp-Asn-Ala-Ser-Y-(X)-Z 3.
(A)-(B)-(X)-Y-Glu-Arg-Tyr-Leu-Lys-Asp-Gln-Gln-Leu- -Leu- (SEQ ID
NO:3) Gly-Ile-Trp-Gly-Cys-Ser-Gly-Lys-Leu-Ile-Y-(X)-- Z 4.
(A)-(B)-(X)-Y-Leu-Gln-Ala-Arg-Ile-Leu-Ala-Val-- Glu-Arg- (SEQ ID
NO:4) Tyr-Leu-Lys-Asp-Gln-Gln-Leu-Y-(X)-Z 5. gp41, isolate Ant70
(A)-(B)-(X)-Y-Leu-Trp-Gly-Cys-Lys-G- ly-Lys-Leu-Val-Cys-Y- (SEQ ID
NO:5) (X)-Z 6. gp41, isolate ELI
(A)-(B)-(X)-Y-Asp-Gln-Gln-Leu-Leu-Gly-Ile-Trp-Gly-Cys-- Ser- (SEQ
ID NO:6) Gly-Lys-His-Ile-Cys-Thr-Thr-Asn-Val-Pro-Trp-Asn- -Y-(X)-Z
b. gp 120 1. Partial V3 loop sequence, consensus
(A)-(B)-(X)-Y-Asn-Asn-Thr-Arg-Lys-Ser-Ile-His-Ile-Gly-Pr- o- (SEQ
ID NO:7) Gly-Arg-Ala-Phe-Tyr-Thr-Thr-Gly-Glu-Ile-Ile-Gly-Y- -(X)-Z
1. a. Complete V3 loop sequence, consensus
(A)-(B)-(X)-Y-Cys-Thr-Arg-Pro-Asn-Asn-Asn-Thr-Arg-Lys-Ser- (SEQ ID
NO:8) Ile-His-Ile-Gly-Pro-Gly-Arg-Ala-Phe-Tyr-Thr-Thr-Gly-Glu-
Ile-Ile-Gly-Asp-Ile-Arg-Gln-Ala-His-Cys-Y-(X)-Z 2. Partial V3 loop
sequence, isolate HIV-1 SF2
(A)-(B)-(X)-Y-Asn-Asn-Thr-Arg-Lys-Ser-Ile-Tyr-Ile-Gly-Pro- (SEQ ID
NO:9) Gly-Arg-Ala-Phe-His-Thr-Thr-Gly-Arg-Ile-Ile-Gly-Y-(X)-Z 3.
Partial V3 loop sequence, isolate HIV-1 SC
(A)-(B)-(X)-Y-Asn-Asn-Thr-Thr-Arg-Ser-Ile-His-Ile-Gly-Pro- (SEQ ID
NO:10) Gly-Arg-Ala-Phe-Tyr-Ala-Thr-Gly-Asp-Ile-Ile-Gly-Y-(X)-Z 4.
Partial V3 loop sequence, isolate HIV-1 MN
(A)-(B)-(X)-Y-Tyr-Asn-Lys-Arg-Lys-Arg-Ile-His-Ile-Gly-Pro- (SEQ ID
NO:11) Gly-Arg-Ala-Phe-Tyr-Thr-Thr-Lys-Asn-Ile-Ile-Gly-Y-(X)-Z 5.
Partial V3 loop sequence, isolate HIV-1 RF
(A)-(B)-(X)-Y-Asn-Asn-Thr-Arg-Lys-Ser-Ile-Thr-Lys-Gly-Pro- (SEQ ID
NO:12) Gly-Arg-Val-Ile-Tyr-Ala-Thr-Gly-Gln-Ile-Ile-Gly-Y-(X)-Z 6.
Partial V3 loop sequence, isolate HIV-1 mal
(A)-(B)-(X)-Y-Asn-Asn-Thr-Arg-Arg-Gly-Ile-His-Phe-Gly-Pro- (SEQ ID
NO:13) Gly-Gln-Ala-Leu-Tyr-Thr-Thr-Gly-Ile-Val-Gly-Y-(X)-Z 7.
Partial V3 loop sequence, isolate HTLV-IIIB
(A)-(B)-(X)-Y-Asn-Asn-Thr-Arg-Lys-Ser-Ile-Arg-Ile-Gln-Arg- (SEQ ID
NO:14) Gly-Pro-Gly-Arg-Ala-Phe-Val-Thr-Ile-Gly-Lys-Ile-Gly-Y-(X)-Z
8. Partial V3 loop sequence, isolate HIV-1 ELI
(A)-(B)-(X)-Y-Gln-Asn-Thr-Arg-Gln-Arg-Thr-Pro-Ile-Gly-Leu- (SEQ ID
NO:15) Gly-Gln-Ser-Leu-Tyr-Thr-Thr-Arg-Ser-Arg-Ser-Y-(X)-Z 9.
Partial V3 loop sequence, isolate ANT70
(A)-(B)-(X)-Y-Gln-Ile-Asp-Ile-Gln-Glu-Met-Arg-Ile-Gly- (SEQ ID
NO:16) Pro-Met-Ala-Trp-Tyr-Ser-Met-Gly-Ile-Gly-Gly-Y-(X)-Z 10.
Partial V3 loop sequence, Brazilian isolate, Peptide V3-368
(A)-(B)-(X)-Y-Asn-Asn-Thr-Arg-Arg-Gly-Ile-His-Met-Gly-Trp- (SEQ ID
NO:17) Gly-Arg-Thr-Phe-Tyr-Ala-Thr-Gly-Glu-Ile-Ile-Gly-Y-(X)-Z 11.
Carboxy-terminus, HIV-1 gp120
(A)-(B)-(X)-Y-Arg-Asp-Asn-Trp-Arg-Ser-Glu-Leu-Tyr-Lys- (SEQ ID
NO:18) Tyr-Lys-Val-Val-Lys-Ile-Glu-Pro-Leu-Gly-Val-Ala-Pro-Thr-
Lys-Ala-Lys-Arg-Arg-Val-Val-Gln-Arg-Glu-Lys-Arg-Y-(X)-Z 2. Human
immunodeficiency Virus type 2 Envelope Peptide a. gp41, isolate
HIV-2 rod (A)-(B)-(X)-Y-Ser-Trp-Gly-Cys-Ala-Phe-Arg--
Gln-Val-Cys-Y- (SEQ ID NO:19) (X)-Z b.
(A)-(B)-(X)-Y-Lys-Tyr-Leu-Gln-Asp-Gln-Ala-Arg-Leu-Asn-Ser- (SEQ ID
NO:20) Trp-Gly-Cys-Ala-Phe-Arg-Gln-Val-Cys-Y-(X)-Z c. gp120,
isolate HIV-2 NiHZ (A)-(B)-(X)-Y-Asn-Lys-Thr-Val-Leu-Pro-Il-
e-Thr-Phe-Met- (SEQ ID NO:21) Ser-Gly-Phe-Lys-Phe-His-Ser-Gln-Pro--
Val-Ile-Asn-Lys-Y-(X)-Z d. Partial V3 loop sequence, Peptide
V3-GB12 (A)-(B)-(X)-Y-Asn-Lys-Thr-Val-Val-Pro-Ile-Thr-Leu--
Met-Ser- (SEQ ID NO:22) Gly-Leu-Val-Phe-His-Ser-Gln-Pro-Ile-Asn-Ly-
s-Y-(X)-Z e. Partial V3 loop sequence, Peptide V3-239
(A)-(B)-(X)-Y-Asn-Lys-Thr-Val-Leu-Pro-Val-Thr-Ile-Met-Ser- (SEQ ID
NO:23) Gly-Leu-Val-Phe-His-Ser-Gln-Pro-Ile-Asn-Asp-Y-(X)-Z 3.
Chimpanzee immunodeficiency Virus a. gp41
(A)-(B)-(X)-Y-Leu-Trp-Gly-Cys-Ser-Gly-Lys-Ala-Val-Cys-Y-(X)-Z (SEQ
ID NO:24) 4. Simian immunodeficiency Virus a. Transmembrane
Protein, isolate SIVagm (TY01)
(A)-(B)-(X)-Y-Ser-Trp-Gly-Cys-Ala-Trp-Lys-Gln-Val-Cys-Y-(X)-Z (SEQ
ID NO:25) b. Transmembrane Protein, isolate SIVmnd
(A)-(B)-(X)-Y-Gln-Trp-Gly-Cys-Ser-Trp-Ala-Gln-Val-Cys-Y-(X)-Z (SEQ
ID NO:26) 5. HTLV-I and HTLV-II Virus Peptide I-gp46-3
(A)-(B)-(X)-Y-Val-Leu-Tyr-Ser-Pro-Asn-Val-Ser-Val-Pro- (SEQ ID
NO:27) Ser-Ser-Ser-Ser-Thr-Leu-Leu-Tyr-Pro-Ser-Leu-Ala-Y-- (X)-Z
Peptide I-gp46-5 (A)-(B)-(X)-Y-Tyr-Thr-Cys-Il-
e-Val-Cys-Ile-Asp-Arg-Ala-Ser- (SEQ ID NO:28)
Leu-Ser-Thr-Trp-His-Val-Leu-Tyr-Ser-Pro-Y-(X)-Z Peptide I-gp46-4
(A)-(B)-(X)-Y-Asn-Ser-Leu-Ile-Leu-Pro-Pro-Phe-Ser-Leu-Ser- - (SEQ
ID NO:29) Pro-Val-Pro-Thr-Leu-Gly-Ser-Arg-Ser-Arg-Arg-Y-(X)- -Z
Peptide I-gp46-6 (A)-(B)-(X)-Y-Asp-Ala-Pro-Gly-T-
yr-Asp-Pro-Ile-Trp-Phe-Leu- (SEQ ID NO:30) Asn-Thr-Glu-Pro-Ser-Gln-
-Leu-Pro-Pro-Thr-Ala-Pro-Pro-Leu-
Leu-Pro-His-Ser-Asn-Leu-Asp-His-I- le-Leu-Glu-Y-(X)-Z Peptide
I-p21-2 (A)-(B)-(X)-Y-Gln-Tyr-Ala-Ala-Gln-Asn-Arg-Arg-Gly-Leu-Asp-
(SEQ ID NO:31)
Leu-Leu-Phe-Trp-Glu-Gln-Gly-Gly-Leu-Cys-Lys-Ala-Leu-Gln-
Glu-Gln-Cys-Arg-Phe-Pro-Y-(X)-Z Peptide I-p19
(A)-(B)-(X)-Y-Pro-Pro-Pro-Pro-Ser-Ser-Pro-Thr-His-Asp-Pro- (SEQ ID
NO:32) Pro-Asp-Ser-Asp-Pro-Gln-Ile-Pro-Pro-Pro-Tyr-Val-Glu-Pro-
Thr-Ala-Pro-Gln-Val-Leu-Y-(X)-Z Peptide II-gp52-1
(A)-(B)-(X)-Y-Lys-Lys-Pro-Asn-Arg-Gln-Gly-Leu-Gly-Tyr-Tyr- (SEQ ID
NO:33) Ser-Pro-Ser-Tyr-Asn-Asp-Pro-Y-(X)-Z Peptide II-gp52-2
(A)-(B)-(X)-Y-Asp-Ala-Pro-Gly-Tyr-Asp-Pro-Leu-Trp-Phe-Il- e- (SEQ
ID NO:34) Thr-Ser-Glu-Pro-Thr-Gln-Pro-Pro-Pro-Thr-Ser-Pro--
Pro-Leu- Val-His-Asp-Ser-Asp-Leu-Glu-His-Val-Leu-Thr-Y-(X)-Z
Peptide II-gp52-3: (A)-(B)-(X)-Y-Tyr-Ser-Cys-Met-Val-Cys--
Val-Asp-Arg-Ser-Ser- (SEQ ID NO:35) Leu-Ser-Ser-Trp-His-Val-Leu-Ty-
r-Thr-Pro-Asn-Ile-Ser-Ile-
Pro-Gln-Gln-Thr-Ser-Ser-Arg-Thr-Ile-Leu-- Phe-Pro-Ser-Y-(X)-Z
Peptide II-p19
(A)-(B)-(X)-Y-Pro-Thr-Thr-Thr-Pro-Pro-Pro-Pro-Pro-Pro-Pro- (SEQ ID
NO:36) Ser-Pro-Glu-Ala-His-Val-Pro-Pro-Pro-Tyr-Val-Glu-Pro-Thr-
Thr-Thr-Gln-Cys-Phe-Y-(X)-Z
[0044] These above-mentioned biotinylated peptides were synthesized
and found to be specifically recognized by antisera from infected
humans or primates are considered particularly advantageous. All
these above-mentioned peptides are new. The process of the
invention enables to increase the antigenicity of these HIV
peptides, which can however be bound to a support, even when they
are not biotinylated.
[0045] The HCV peptide sequences, which follow, have been found to
be specifically recognized by antisera from infected humans or
primates and which are considered particularly advantageous. The
non-biotinylated amino acid sequences can be synthesized according
to classical methods.
[0046] The peptides of interest are intended to mimic
immunologically proteins or domains of proteins encoded by HCV.
Since sequence variability has been observed for HCV, it may be
desirable to vary one or more amino acids so as to better mimic the
epitopes of different strains. It should be understood that the
peptides described need not be identical to any particular HCV
sequence as long as the subject compounds are capable of providing
for immunological competition with at least one strain of HCV.
[0047] The peptides may therefore be subject to insertions,
deletions and conservative as well as non-conservative amino acid
substitutions where such changes might provide for certain
advantages in their use. The peptides will preferably be as short
as possible while still maintaining all of the sensitivity of the
larger sequence. In certain cases, it may be desirable to join two
or more peptides together into a single structure.
[0048] The formation of such a composite may involve covalent or
non-covalent linkages.
[0049] Of particular interest are biotinylated peptides of HCV into
which cysteine, thioglycollic acid, or other thiol-containing
compounds have been incorporated into the peptide chain for the
purpose of providing mercapto-groups, which can be used, for
cyclization of the peptides.
[0050] The following peptides from the Core region of HCV were
determined as corresponding to immunologically important
epitopes.
[0051] 1. Peptide I or Core I (aa. 1-20) has the following amino
acid sequence:
2 (I) (A)-(B)-(X)-Y-Met-Ser-Thr-Ile-Pro-Lys-Pro-Gln-Arg-Ly- s- (SEQ
ID NO:37) Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-- Y-(X)-Z
[0052] 2. Peptide II or Core 2 (aa. 7-26) has the amino acid
sequence:
3 (II) (A)-(B)-(X)-Y-Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-A- sn-
(SEQ ID NO:38) Arg-Arg-Pro-Gln-Asp-Val-Lys-Phe-Pro-Gly-
-Y-(X)-Z
[0053] Of particular interest is the oligopeptide IIA (aa. 8 to
18):
4 (IIA) (A)-(B)-(X)-Y-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn--
Arg-Arg- (SEQ ID NO:39) Y-(X)-Z
[0054] 3. Peptide III or Core 3 (aa 13-32) has the sequence:
5 (III) (A)-(B)-(X)-Y-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Asp-- Val-
(SEQ ID NO:40) Lys-Phe-Pro-Gly-Gly-Gly-Gln-Ile-Val-Gl-
y-Y-(X)-Z
[0055] 4. Peptide IV or Core 7 (aa 37-56) has the sequences:
6 (IV) (A)-(B)-(X)-Y-Leu-Pro-Arg-Arg-Gly-Pro-Arg-Leu-Gly-V- al-
(SEQ ID NO:41) Arg-Ala-Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser-
-Y-(X)-Z
[0056] Of particular interest is the oligopeptide. IVa or Core 6
(aa. 31 to 50):
7 (IVa) (A)-(B)-(X)-Y-Val-Gly-Gly-Val-Tyr-Leu-Leu-Pro-Arg-- Arg-
(SEQ ID NO:42) Gly-Pro-Arg-Leu-Gly-Val-Arg-Ala-Thr-Ar-
g-Y-(X)-Z
[0057] 5. Peptide V or Core 9 (aa 49-68) has the sequence:
8 (V) (A)-(B)-(X)-Y-Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser-Gln-Pr- o- (SEQ
ID NO:43) Arg-Gly-Arg-Arg-Gln-Pro-Ile-Pro-Lys-Val-- Y-(X)-Z
[0058] Of particular interest is the oligopeptide Va (aa. 55 to
74):
9 (Va) (A)-(B)-(X)-Y-Arg-Ser-Gln-Pro-Arg-Gly-Arg-Gln-Pro- (SEQ ID
NO:44) Ile-Pro-Lys-Val-Arg-Arg-Pro-Glu-Gly-Arg-Y-- (X)-Z
[0059] 6. Peptide VI or Core 11 (aa 61-80) has the following
sequence:
10 (VI) (A)-(B)-(X)-Y-Arg-Arg-Gln-Pro-Ile-Pro-Lys-Val-Arg-- Arg-
(SEQ ID NO:45) Pro-Glu-Gly-Arg-Thr-Trp-Ala-Gln-Pro-Gl-
y-Y-(X)-Z
[0060] 7. Peptide VII (aa 73-92) or core 13 has the sequence:
11 (VII) (A)-(B)-(X)-Y-Gly-Arg-Thr-Trp-Ala-Gln-Pro-Gly-Tyr-Pro-
(SEQ ID NO:46) Trp-Pro-Leu-Tyr-Gly-Asn-Glu-Gly-Cys-Gly--
y-(X)-Z
[0061] 8. Peptide Core 123 (aa. 1-32):
12 (A)-(B)-(X)-Y-Met-Ser-Thr-Ile-Pro-Gln-Arg-Lys-Thr-Lys- (SEQ ID
NO:47) Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Asp-Val-Lys-Phe-P- ro-Gly-
Gly-Gly-Gln-Ile-Val-Gly-Y-(X)-Z
[0062] 9. Peptide Core 7910 (aa. 37-80):
13 (A)-(B)-(X)-Y-Gly-Gly-Val-Tyr-Leu-Leu-Pro-Arg-Arg-Gly-Pro- (SEQ
ID NO:48) Arg-Leu-Gly-Val-Arg-Arg-Ala-Thr-Arg-Lys-Thr-Se-
r-Glu-Arg- Ser-Gln-Pro-Arg-Gly-Arg-Arg-Gln-Pro-Ile-Pro-Lys-
-Val-Arg- Arg-Y-(X)-Z
[0063] The following peptides from the NS4 region of HCV were found
to correspond to immunologically important epitopes.
[0064] Peptide VIII or NS4-1 or HCV1 (aa 1688-1707) has the
sequence:
14 (VIII) (A)-(B)-(X)-Y-Leu-Ser-Gly-Lys-Pro-Ala-Ile-Ile-Pro-Asp-
(SEQ ID NO:49) Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu--
Y-(X)-Z
[0065] Peptide IX or HCV2 (aa 1694-1713) has the sequence:
15 (IX) (A)-(B)-(X)-Y-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg- (SEQ
ID NO:50) Glu-Phe-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-Y-(X)-Z Peptide
HCV3 (A)-(B)-(X)-Y-Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Met-G- lu-Glu-
(SEQ ID NO:51) Cys-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Y-(X)-Z
[0066] Peptide X or HCV4 (aa 1706-1725) has the sequence:
16 (X) (A)-(B)-(X)-Y-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-His-Leu- (SEQ
ID NO:52) Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Y-(X)- -Z
[0067] 11. Peptide XI or NS4-5 or HCV5 (aa 1712-1731) has the
sequence:
17 (XI) (A)-(B)-(X)-Y-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly- (SEQ
ID NO:53) Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Eys-Y-(X)- -Z
[0068] 12. Peptide XII or HCV6 (aa 1718-1737) has the sequence:
18 (XII) (A)-(B)-(X)-Y-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-
(SEQ ID NO:54) Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln--
Y-(X)-Z
[0069] 13. Peptide XIII or NS4-7 or HCV7 (aa 1724-1743) has the
sequence:
19 (XIII) (A)-(B)-(X)-Y-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-
(SEQ ID NO:55) Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala--
Y-(X)-Z
[0070] 14. Peptide XIV or HCV8 (aa 1730-1749) has the sequence:
20 (XIV) (A)-(B)-(X)-Y-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-
(SEQ ID NO:56) Ser-Arg-Gln-Ala-Glu-Val-Ile-Ala-Pro-Ala--
Y-(X)-Z
[0071] 15. Peptide NS4-27 or HCV9 (aa. 1712-1743):
21 (A)-(B)-(X)-Y-Ser-Gln-His-Leu-Pro- (SEQ ID NO:57)
Tyr-Ile-Glu-Gln-Glu-Met-Leu-Ala-Glu-
Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-
Leu-Gln-Thr-Ala-Ser-Arg-Gln-Ala-Y- (X)-Z
[0072] 16. Peptide. NS4e:
22 (A)-(B)-(X)-Y-Gly-Glu-Gly-Ala-Val- (SEQ ID NO:58)
Gln-Trp-Met-Asn-Arg-Leu-Ile-Ala-Phe-
Ala-Ser-Arg-Gly-Asn-His-Y-(X)-Z
[0073] The following peptides of the NS5 region of HCV were found
to correspond to immunologically important epitopes.
[0074] Peptide XV or NS5-25 (aa 2263-2282) has the sequence:
23 (XV) (A)-(B)-(X)-Y-Glu-Asp-Glu-Arg-Glu- (SEQ ID NO:59)
Ile-Ser-Val-Pro-Ala-Glu-Ile-Leu-Arg-
Lys-Ser-Arg-Arg-Phe-Ala-Y-(X)-Z
[0075] Peptide XVI or NS5-27 (aa 2275-2294) has the sequence:
24 (XVI) (A)-(B)-(X)-Y-Leu-Arg-Lys-Ser-Arg- (SEQ ID NO:60)
Arg-Phe-Ala-Gln-Ala-Leu-Pro-Val'Trp-
Ala-Arg-Pro-Asp-Tyr-Asn-Y-(X)-Z
[0076] Peptide XVII or NS5-29 (aa 2287-2306) has the sequence:
25 (XVII) (A)-(B)-(X)-Y-Val-Trp-Ala-Arg-Pro- (SEQ ID NO:61)
Asp-Tyr-Asn-Pro-Pro-Leu-Val-Glu-Thr-
Trp-Lys-Lys-Pro-Asp-Tyr-Y-(X)-Z
[0077] Peptide XVIII or NS5-31 (aa 2299-2318) has the sequence:
26 (XVIII) (A)-(B)-(X)-Y-Glu-Thr-Trp-Lys-Lys- (SEQ ID NO:62)
Pro-Asp-Tyr-Glu-Pro-Pro-Val-Val-His-
Gly-Cys-Pro-Leu-Pro-Pro-Y-(X)-Z
[0078] Peptide XIX or NS5-33 (aa 2311-2330) has the sequence:
27 (XIX) (A)-(B)-(X)-Y-Val-His-Gly-Cys-Pro- (SEQ ID NO:63)
Leu-Pro-Pro-Pro-Lys-Ser-Pro-Pro-Val-
Pro-Pro-Pro-Arg-Lys-Lys-Y-(X)-Z
[0079] Peptide NS5-2527 (aa. 2263 to 2294):
28 (A)-(B)-(X)-Y-Glu-Asp-Glu-Arg-Glu- (SEQ ID NO:64)
Ile-Ser-Val-Pro-Ala-Glu-Ile-Leu-Arg-
Lys-Ser-Arg-Lys-Ser-Arg-Arg-Phe-Ala-
Gln-Ala-Leu-Pro-Val-Trp-Ala-Arg-Pro-
Asp-Tyr-Asp-Tyr-Asn-Y-(X)-Z
[0080] The following peptides from the N-terminal region of the
E2/NS1 region of HCV were found to correspond to immunologically
important epitopes.
29 Peptide XXa (aa. 383-416) (A)-(B)-(X)-Y-Gly-Glu-Thr-Tyr- -Thr-
(SEQ ID NO:65) Ser-Gly-Gly-Ala-Ala-Ser-His-Thr-Thr-
Ser-Thr-Leu-Ala-Ser-Leu-Phe-Ser-Pro-
Gly-Ala-Ser-Gln-Arg-Ile-Gln-Leu-Val- Asn-Thr-Y-(X)-Z Peptide XXa-1
(aa. 383-404) (A)-(B)-(X)-Y-Gly-Glu-Thr-T- yr-Thr- (SEQ ID NO:66)
Ser-Gly-Gly-Ala-Ala-Ser-His-Thr-Thr- -
Ser-Thr-Leu-Ala-Ser-Leu-Phe-Ser-Y- (X)-Z Peptide XXa-2 (aa.
393-416) (A)-(B)-(X)-Y-Ser-His-Thr-Thr-Ser- (SEQ ID NO:67)
Thr-Leu-Ala-Ser-Leu-Phe-Ser-Pro-Gly-
Ala-Ser-Gln-Arg-Ile-Gln-Leu-Val-Asn- Thr-Y-(X)-Z Peptide XXb (aa.
383-416) (A)-(B)-(X)-Y-Gly-His-Thr-Arg-Val- - (SEQ ID NO:68)
Ser-Gly-Gly-Ala-Ala-Ala-Ser-Asp-Thr-
Arg-Gly-Leu-Val-Ser-Leu-Phe-Ser-Pro-
Gly-Ser-Ala-Gln-Lys-Ile-Gln-Leu-Val- Asn-Thr-Y-(X)-Z Peptide XXb-1
(aa. 383-404) (A)-(B)-(X)-Y-Gly-His-Thr-A- rg-Val- (SEQ ID NO:69)
Ser-Gly-Gly-Ala-Ala-Ala-Ser-Asp-Thr- -
Arg-Gly-Leu-Val-Ser-Leu-Phe-Ser-Y- (X)-Z Peptide XXb-2 (aa.
393-416) (A)-(B)-(X)-Y-Ala-Ser-Asp-Thr-Arg- (SEQ ID NO:70)
Gly-Leu-Val-Ser-Leu-Phe-Ser-Pro-Gly-
Ser-Ala-Gln-Lys-Ile-Gln-Leu-Val-Asn- Thr-Y-(X)-Z Peptide XXc (aa.
383-416) (A)-(B)-(X)-Y-Gly-His-Thr-Arg-Val- - (SEQ ID NO:71)
Thr-Gly-Gly-Val-Gln-Gly-His-Val-Thr-
Cys-Thr-Leu-Thr-Ser-Leu-Phe-Arg-Pro-
Gly-Ala-Ser-Gln-Lys-Ile-Gln-Leu-Val- Asn-Thr-Y-(X)-Z Peptide XXc-1
(aa. 383-404) (A)-(B)-(X)-Y-Gly-His-Thr-A- rg-Val- (SEQ ID NO:72)
Thr-Gly-Gly-Val-Gln-Gly-His-Val-Thr- -
Cys-Thr-Leu-Thr-Ser-Leu-Phe-Arg-Y- (X)-Z Peptide XXc-2 (aa.
393-416) (A)-(B)-(X)-Y-Gly-His-Val-Thr-Cys- (SEQ ID NO:73)
Thr-Leu-Thr-Ser-Leu-Phe-Arg-Pro-Gly-
Ala-Ser-Gln-Lys-Ile-Gln-Leu-Val-Asn- Thr-Y-(X)-Z Peptide XXd (aa.
383-416) (A)-(B)-(X)-Y-Gly-His-Thr-His-Val- - (SEQ ID NO:74)
Thr-Gly-Gly-Arg-Val-Ala-Ser-Ser-Thr-
Gln-Ser-Leu-Val-Ser-Trp-Leu-Ser-Gln-
Gly-Pro-Ser-Gln-Lys-Ile-Gln-Leu-Val- Asn-Thr-Y-(X)-Z Peptide XXd-1
(aa. 383-404) (A)-(B)-(X)-Y-Gly-His-Thr-H- is-Val- (SEQ ID NO:75)
Thr-Gly-Gly-Arg-Val-Ala-Ser-Ser-Thr- -
Gln-Ser-Leu-Val-Ser-Trp-Leu-Ser-Y- (X)-Z Peptide XXd-2 (aa.
393-416) (A)-(B)-(X)-Y-Ala-Ser-Ser-Thr-Gln- (SEQ ID NO:76)
Ser-Leu-Val-Ser-Trp-Leu-Ser-Gln-Gly-
Pro-Ser-Gln-Lys-Ile-Gln-Leu-Val-Asn- Thr-Y-(X)-Z Peptide XXe (aa.
383-416) (A)-(B)-(X)-Y-Gly-Asp-Thr-His-Val- - (SEQ ID NO:77)
Thr-Gly-Gly-Ala-Gln-Ala-Lys-Thr-Thr-
Asn-Arg-Leu-Val-Ser-Met-Phe-Ala-Ser-
Gly-Pro-Ser-Gln-Lys-Ile-Gln-Leu-Ile- Asn-Thr-Y-(X)-Z Peptide XXe-1
(aa. 383-404) (A)-(B)-(X)-Y-Gly-Asp-Thr-H- is-Val- (SEQ ID NO:78)
Thr-Gly-Gly-Ala-Gln-Ala-Lys-Thr-Thr- -
Asn-Arg-Leu-Val-Ser-Met-Phe-Ala-Y- (X)-Z Peptide XXe-2 (aa.
393-416) (A)-(B)-(X)-Y-Ala-Lys-Thr-Thr-Asn- (SEQ ID NO:79)
Arg-Leu-Val-Ser-Met-Phe-Ala-Ser-Gly-
Pro-Ser-Gln-Lys-Ile-Gln-Leu-Ile-Asn- Thr-Y-(X)-Z Peptide XXf (aa.
383-416) (A)-(B)-(X)-Y-Ala-Glu-Thr-Tyr-Thr- - (SEQ ID NO:80)
Ser-Gly-Gly-Asn-Ala-Gly-His-Thr-Met-
Thr-Gly-Ile-Val-Arg-Phe-Phe-Ala-Pro-
Gly-Pro-Lys-Gln-Asn-Val-His-Leu-Ile- Asn-Thr-Y-(X)-Z Peptide XXf-1
(aa. 383-404) (A)-(B)-(X)-Y-Ala-Glu-Thr-T- yr-Thr- (SEQ ID NO:81)
Ser-Gly-Gly-Asn-Ala-Gly-His-Thr-Met- -
Thr-Gly-Ile-Val-Arg-Phe-Phe-Ala-Y- (X)-Z Peptide XXf-2 (aa.
393-416) (A)-(B)-(X)-Y-Gly-His-Thr-Met-Thr- (SEQ ID NO:82)
Gly-Ile-Val-Arg-Phe-Phe-Ala-Pro-Gly-
Pro-Lys-Gln-Asn-Val-His-Leu-Ile-Asn- Thr-Y-(X)-Z Peptide XXg (aa.
383-416) (A)-(B)-(X)-Y-Ala-Glu-Thr-Ile-Val- - (SEQ ED NO:83)
Ser-Gly-Gly-Gln-Ala-Ala-Arg-Ala-Met-
Ser-Gly-Leu-Val-Ser-Leu-Phe-Thr-Pro-
Gly-Ala-Lys-Gln-Asn-Ile-Gln-Leu-Ile- Asn-Thr-Y-(X)-Z Peptide XXg-1
(aa. 383-404) (A)-(B)-(X)-Y-Ala-Glu-Thr-I- le-Val- (SEQ ID NO:84)
Ser-Gly-Gly-Gln-Ala-Ala-Arg-Ala-Met- -
Ser-Gly-Leu-Val-Ser-Leu-Phe-Thr-Y- (X)-Z Peptide XXg-2 (aa.
393-416) (A)-(B)-(X)-Y-Ala-Arg-Ala-Met-Ser- (SEQ ID NO:85)
Gly-Leu-Val-Ser-Leu-Phe-Thr-Pro-Gly-
Ala-Lys-Gln-Asn-Ile-Gln-Leu-Ile-Asn- Thr-Y-(X)-Z Peptide XXh (aa.
383-416) (A)-(B)-(X)-Y-Ala-Glu-Thr-Tyr-Thr- - (SEQ ID NO:86)
Thr-Gly-Gly-Ser-Thr-Ala-Arg-Thr-Thr-
Gln-Gly-Leu-Val-Ser-Leu-Phe-Ser-Arg-
Gly-Ala-Lys-Gln-Asp-Ile-Gln-Leu-Ile- Asn-Thr-Y-(X)-Z Peptide XXh-1
(aa. 383-404) (A)-(B)-(X)-Y-Ala-Glu-Thr-T- yr-Thr- (SEQ ID NO:87)
Thr-Gly-Gly-Ser-Thr-Ala-Arg-Thr-Thr- -
Gln-Gly-Leu-Val-Ser-Leu-Phe-Ser-Y- (X)-Z Peptide XXh-2 (aa.
393-416) (A)-(B)-(X)-Y-Ala-Arg-Thr-Thr-Gln- (SEQ ID NO:88)
Gly-Leu-Val-Ser-Leu-Phe-Ser-Arg-Gly-
Ala-Lys-Gln-Asp-Ile-Gln-Leu-Ile-Asn- Thr-Y-(X)-Z
[0081] The above-mentioned sequences correspond to epitopes
localized on the HCV type-1 isolate HCV-1 (Choo et al. Proc; Natl.
Acad. Sci. 88, 2451-2455, 1991) and HC-J1 (Okamoto et al., Jap. J.
Exp. Med. 60, 167-177, 1990) sequence. It is, however, to be
understood that also peptides from other type-1 HCV isolate
sequences which correspond to the above-mentioned immunologically
important regions may also be comprised in the composition
according to the invention. An example of variant HCV sequences
also falling within the present invention may be derived from the
HCV-J isolate (Kato et al., Proc. Natl. Acad. Sci. 87,
9524-9528).
[0082] The following peptides derived from the same regions as the
above-cited peptide regions from the type 2 HCV sequences.
30 Peptide XX/2 (A)-(B)-(X)-Y-Ala-Gln-Thr-His-Thr- (SEQ ID NO:89)
Val-Gly-Gly-Ser-Thr-Ala-His-Asn- Ala-Arg-Thr-Leu-Thr-Gly-Met-Phe-
Ser-Leu-Gly-Ala-Arg-Gln-- Lys-Ile- Gln-Leu-Ile-Asn-Thr-Y-(X)-Z
Peptide XX/2-1 (A)-(B)-(X)-Y-Ala-Gln-Thr-His-Thr- (SEQ ID NO:90)
Val-Gly-Gly-Ser-Thr-Ala-His-Asn- Ala-Arg-Thr-Leu-Thr-Gly-Met-Phe-
Ser-Y-(X)-Z Peptide XX/2-2 (A)-(B)-(X)-Y-Ala-His-Asn-Ala-Arg- (SEQ
ID NO:91) Thr-Leu-Thr-Gly-Met-Phe-Ser-Leu-
Gly-Ala-Arg-Gln-Lys-Ile-Gln-Leu- Ile-Asn-Thr-Y-(X)-Z Peptide VIII-2
or NS4-1 (2) (A)-(B)-(X)-Y-Val-Asn-Gln-A- rg-Ala- (SEQ ID NO:92)
Val-Val-Ali-Pro-Asp-Lys-Glu-Val- Leu-Tyr-Glu-Ala-Phe-Asp-Glu-Y-(X)-
Z Peptide IX-2 (A)-(B)-(X)-Y-Val-Ala-Pro-Asp-Lys- (SEQ ID NO:93)
Glu-Val-Leu-Tyr-Glu-Ala-Phe-Asp- Glu-Met-Glu-Glu-Cys-Ala-Ser-Y-(X)-
Z Peptide X-2 (A)-(B)-(X)-Y-Asp-Glu-Met-Glu-Glu- (SEQ ID NO:94)
Cys-Ala-Ser-Arg-Ala-Ala-Leu-Ile- Glu-Glu-Gly-Gln-Arg-Ile-Ala-Y-(X)-
Z Peptide XI-2 or NS4-5 (2) (A)-(B)-(X)-Y-Ala-Ser-Arg-Ala-Ala- (SEQ
ID NO:95) Leu-Ile-Glu-Glu-Gly-Gln-Arg-Ile-
Ala-Glu-Met-Leu-Lys-Ser-Lys-Y-(X)- Z Peptide XII-2
(A)-(B)-(X)-Y-Ile-Glu-Glu-Gly-Gln- (SEQ ID NO:96)
Arg-Ile-Ala-Glu-Met-Leu-Lys-Ser- Lys-Ile-Gln-Gly-Leu-Leu-Gln-Y-(X)-
Z Peptide XIII-2 or NS4-7 (2) (A)-(B)-(X)-Y-Ile-Ala-Glu-Met-Leu-
(SEQ ID NO:97) Lys-Ser-Lys-Ile-Gln-Gly-Leu-Leu-
Gln-Gln-Ala-Ser-Lys-Gln-Ala-Y-(X)- Z Peptide XIV-2
(A)-(B)-(X)-Y-Ser-Lys-Ile-Gln-Gly- (SEQ ID NO:98)
Leu-Leu-Gln-Gln-Ala-Ser-Lys-Gln- Ala-Gln-Asp-Ile-Gln-Pro-Ala-Y-(X)-
Z Peptide XV-2 (A)-(B)-(X)-Y-Arg-Ser-Asp-Leu-Glu- (SEQ ID NO:99)
Pro-Ser-Ile-Pro-Ser-Glu-Tyr-Met- Leu-Pro-Lys-Lys-Arg-Phe-Pro-(X)-Y-
Z Peptide XVI-2 (A)-(B)-(X)-Y-Met-Leu-Pro-Lys-Lys- (SEQ ID NO:100)
Arg-Phe-Pro-Pro-Ala-Leu-Pro-Ala- Trp-Ala-Arg-Pro-Asp-Tyr-Asn-Y-(X)-
Z Peptide XVII-2 (A)-(B)-(X)-Y-Ala-Trp-Ala-Arg-Pro- (SEQ ID NO:101)
Asp-Tyr-Asn-Pro-Pro-Leu-Val-Glu- Ser-Trp-Lys-Arg-Pro-Asp-Tyr-Y-(X)-
Z Peptide XVIII-2 (A)-(B)-(X)-Y-Glu-Ser-Trp-Lys-Arg- (SEQ ID
NO:102) Pro-Asp-Tyr-Gln-Pro-Ala-Thr-Val-
Ala-Gly-Cys-Ala-Leu-Pro-Pro-Y-(X)- Z Peptide XIX-2
(A)-(B)-(X)-Y-Val-Ala-Gly-Cys-Ala- (SEQ ID NO:103)
Leu-Pro-Pro-Pro-Lys-Lys-Thr-Pro- Thr-Pro-Pro-Pro-Arg-Arg-Arg-Y-(X)-
Z
[0083] The above-mentioned sequences correspond to epitopes
localized on the HCV type-2 isolate HC-J6 sequence (Okamoto et al.,
J. Gen. Virology 72, 2697-2704, 1991). It is however, to be
understood that also peptides from other type-2 HCV isolate
sequences which correspond to the above-mentioned immunologically
important regions may also be comprised in the composition
according to the invention.
[0084] Examples of variant sequences also falling within the
present invention may be derived from HCV isolate HC-J8 (Okamato et
al., Virology 188, 331-341, 1992).
[0085] The following peptides from the NS4 region of HCV type 3 are
also preferred peptides according to the; present invention:
31 Peptide NS4-1 (3) (A)-(B)-(X)-Y-Leu-Gly-Gly-Lys-Pro- (SEQ ID
NO:107) Ala-Ile-Val-Pro-Asp-Lys-Glu-Val-
Leu-Tyr-Gln-Gln-Tyr-Asp-Glu-Y-(X)- Z Peptide NS4-5 (3)
(A)-(B)-(X)-Y-Ser-Gln-Ala-Ala-Pro- (SEQ ID NO:108)
Tyr-Ile-Glu-Gln-Ala-Gln-Val-Ile- Ala-His-Gln-Phe-Lys-Glu-Lys-Y-(X)-
Z Peptide NS4-7 (3) (A)-(B)-(X)-Y-Ile-Ala-His-Gln-His- (SEQ ID
NO:109) Gln-Phe-Lys-Glu-Lys-Val-Leu-Gly-
Leu-Leu-Gln-Arg-Ala-Thr-Gln-Gln- Gln-Y-(X)-Z
[0086] It is to be understood that also other peptides
corresponding to HCV type-3 isolate sequences which YAC). 13/18054
PCF/EP93/00517 correspond to immunologically important regions as
determined for HCV type-1 and type-2 may also be comprised in the
composition according to the invention.
[0087] The composition according to the present invention may also
comprise hybrid HCV peptide sequences consisting of combinations of
the core epitopes of the HCV core (table 9) HCV NS4 (table 10) or
the HCV NS5 (table 11) region separated by Gly and/or Ser residues,
and preferentially the following hybrid HCV sequences:
32 Epi-152 (A)-(B)-(X)-Y-Ile-Pro-Asp-Arg-Glu- (SEQ ID NO:104)
Val-Leu-Tyr-Arg-Gly-Gly-Lys-Lys- Pro-Asp-Tyr-Glu-Pro-Pro-Val-Gly-
Gly-Arg-Arg-Pro-Gln-Asp-- Val-Lys- Phe-Pro-Y-(X)-Z Epi-33B3A
(A)-(B)-(X)-Y-Trp-Ala-Arg-Pro-Asp- (SEQ ID NO:105)
Tyr-Asn-Pro-Pro-Gly-Gly-Gln-Phe- Lys-Gln-Lys-Ala-Leu-Gly-- Leu-Gly-
Ser-Gly-Val-Tyr-Leu-Leu-Pro-Arg- Arg-Gly-Y-(X)-Z Epi-4B2A6
(A)-(B)-(X)-Y-Arg-Gly-Ar- g-Arg-Gln- (SEQ ID NO:106)
Pro-Ile-Pro-Lys-Gly-Gly-Ser-Gln- - His-Leu-Pro-Tyr-Ile-Glu-Gln-Ser-
Gly-Pro-Val-Val-His-Gly-Cys-Pro- Leu-Pro-Y-(X)-Z
[0088] The composition according to the present invention may also
comprise so called biotinylated mixotope sequences consisting of
peptides containing at each position all the amino acids found in
the naturally occurring isolates, with said peptides being derived
from any of the above-mentioned immunologically important regions
(see FIG. 14).
[0089] (2) A preferred mixture of biotinylated peptides for
detecting and/or immunizing against Hepatitis C Virus, Human
Immunodeficiency Virus type 1 and human Immunodeficiency Virus type
2 consists of:
[0090] A. II, III, IVa, Va, IX, XI, XIII, XV, XVI, XVIII,
[0091] 1a.3, 1a.4, 1a.b, 1b.1a, 2b, 2d,
[0092] B. II, III, IVa, Va, IX, IX-2, XI, XI-2, XIII, XIII-2, XV,
XV-2, XVI, XVI-2, XVIII, XVIII-2,
[0093] 1a.3, 1a.4, 1a.b, 1b.1a, 2b, 2d.
[0094] (3) A preferred mixture of biotinylated peptides for
detecting and/or immunizing against Human Immunodeficiency Virus
types 1 and 2 and Human Lymphotropic Virus types I and II consists
of:
[0095] 1a.3, 1a.4, 1b.1, 2b, 2c, 2d, I-gp46-3, I-gp46-4, I-gp46-5,
I-gp46-6, II-gp52-2, II-gp52-3, I-p21-2, I-p19, II-p19.
[0096] (4) Another preferred mixture of biotinylated peptides for
detecting and/or immunizing against Hepatitis C Virus, Human
Immunodeficiency Virus types 1 and 2 and Human Lymphotropic virus
types I and II consists of:
[0097] 1a.3, 1a.4, 1a.6, 1b.1a, 2d, II, III, IVa, Va, IX, XI, XIII,
XV, XVI, XVIII, XXa-2, XXc-2, XXg-2, XXh-2, I-gp46-3, I-gp46-4,
I-gp46-5, I-gp46-6, II-gp52-3, I-p21-2, I-P19, II-P19.
[0098] (5) The present invention relates also to compositions of
biotinylated peptides which are considered particularly
advantageous, for diagnostic as well as immunogenic purpose for
Hepatitis C Virus, and which advantageously comprise the following
mixtures:
[0099] A. I, III, IVa, Va,
[0100] B. II, III, IVa, Va,
[0101] C. IX, XI, XIII,
[0102] D. XV, XVI, XVIII, XIX,
[0103] E. XXc-2, XXa-1, XXa-2, XXh-1, XXh-2, XXg-2, XX/2-2,
[0104] F. IX-2, XI-2, XIII-2,
[0105] G. XV-2, XVI-2, XVIII-2, XIX-2,
[0106] H. IX, IX-2, XI, XI-2, XIII, XIII-2,
[0107] I. XV, XV-2, XVI, XVI-2, XVIII, XVIII-2, XIX, XIX-2,
[0108] J. II, III, IVa, Va, IX, IX-2, XI, XI-2, XIII, XIII-2, XV,
XV-2, XVI, XVI-2, XVIII, XVIII-2,
[0109] K. II, III, IVa, Va, IX, XI, XIII, XV, XVI, XVIII,
[0110] L. II, III, IV, V, IX, XI, XIII, XV, XVI, XVIII,
[0111] M. II, III, IVa, Va, IX, XI, XIII, XV, XVI, XVIII, XXa-2,
XXc-2, XXg-2, XXh-2.
[0112] (6) The present invention relates also to compositions of
biotinylated peptides which are considered particularly
advantageous, for diagnostic as well as immunogenic purposes for
Human Immunodeficiency virus, and which are advantageously selected
from the following mixtures:
[0113] For type 1:
[0114] A. 1a.3, 1a.4, 1a.5, 1a.b
[0115] B. 1a.3, 1a.4, 1b.1, 1b.3, 1b.6, 1b.10,
[0116] C. 1b.1, 1b.2, 1b.3, 1b.4, 1b.5, 1b.6, 1b.7, 1b.8, 1b.9,
1b.10
[0117] D. 1b.1, 1b.2, 1b.3, 1b.4, 1b.6, 1b.10,
[0118] E. 1a.3, 1a.4, 1a.5, 1a.b, 1b.1a.
[0119] For type 2:
[0120] A. 2b, 2c, 2d, 2e.
[0121] For types 1 and 2:
[0122] A. 1a.3, 1a.4, 1b.1, 2b, 2c, 2d,
[0123] B. 1a.3, 1a.4, 1b.1a, 2b, 2d.
[0124] (7) The present invention relates also to compositions
comprising biotinylated peptides, which are considered particularly
advantageous, for diagnostic as well as immunogenic purposes for
Human T-cell Lymphotropic virus and are advantageously, selected
from the following mixtures:
[0125] For Human T-Lymphotropic virus type I:
[0126] Peptides I-gp46-3, I-gp46-4, I-gp46-5, I-gp46-6, I-p21-2,
I-p19
[0127] For Human T-Lymphotropic virus type II
[0128] Peptides II-gp52-1, II-gp52-2, II-gp52-3, I-gp46-4, II-p19,
I-p21-2.
[0129] For Human lymphotropic virus types I and II:
[0130] Peptides I-qp46-3, I-qp46-4, I-gp46-5, I-gp46-6, II-gp52-1,
IIgp52-2, II-gp52-3, I-p21-2, I-P19, II-P19.
[0131] The synthesis of the peptides may be achieved in solution or
on a solid support. Synthesis protocols generally employ
t-butyloxycarbonyl- or 9-fluorenylmethoxycarbonyl-protected
activated amino acids.
[0132] The procedures for carrying out the synthesis, the amino
acid activation techniques, the types of side-chain production, and
the cleavage procedures used are amply described in, for example,
Stewart and Young, Solid Phase Peptide Synthesis, 2nd Edition,
Pierce; Chemical Company, 1984; and Atherton and Sheppard, Solid
Phase Peptide Synthesis, IRL Press, 1989.
[0133] (8) The present invention also relates to a process for in
vitro determination of antibodies using the above defined
biotinylated peptides, wherein said biotinylated peptides are
preferably, in the form of streptavidin-biotinylated peptide
complexes or avidin-biotinylated peptide complexes.
[0134] In the complex of streptavidin-biotinylated peptides or
avidin-biotinylated peptides, the peptides may be biotinylated
either N-terminally, C-terminally or internally.
[0135] This approach for the determination of antibodies is not
limited with respect to peptide length and avoids the difficulties
inherent in coating peptides directly onto the solid phase for
immunological evaluation.
[0136] The use of biotinylated peptides, in the process of the
invention, makes the anchorage of peptides to a solid support such
that it leaves their essential amino acids free to be recognized by
antibodies.
[0137] The expression anchoring peptide to a solid support means
the attachment of the peptide to a support via covalent bonds or
non-covalent interactions such that the peptide becomes
immobilized.
[0138] The solid support can be nitrocellulose, polystyrene, nylon
or any other natural or synthetic polymer.
[0139] The expression "their essential amino acids are left free to
be recognized by antibodies" means that amino acid side chains of
the peptide proper are neither chemically modified in any way nor
involved in the interaction between the peptide and the solid
phase.
[0140] The use of biotinylated peptides in the process of the
invention enables said biotinylated peptides to be free to assume a
wide range of conformations, among which at least one is
appropriate for the binding of antibodies to said biotinylated
peptides.
[0141] Any biotinylated peptide can be selected to be used in the
process of the invention. However, some of them are able to be
anchored on solid support and to react with antibodies specifically
recognizing the epitope-within this peptide even without being
biotinylated and without being involved in a complex of avidin of
streptavidin. In this case, the use of biotinylated peptides
results in an apparent increase of the antigenicity of peptides
with respect to the antigenicity observed when the peptides are not
biotinylated. The expression "apparent" is meant to indicate an
observed change obtained under similar test conditions without
regard to the absolute cause of the observed change.
[0142] By "antigenicity" is meant the property of a peptide to be
bound by an antibody.
[0143] By "increase of antigenicity" is meant that a positive
signal is obtained for a dilution which is at least two times the
dilution of the non-biotinylated peptides. Said positive signal is
of the same magnitude as the one obtained for non-biotinylated
peptides.
[0144] In other words, obtaining a positive signal can be obtained
for a smaller amount of biotinylated peptide, compared to the
amount of non-biotinylated peptide.
[0145] The present invention also illustrated a process for the
identification of epitopes in a protein sequence comprises the
following steps:
[0146] the preparation of peptides corresponding to portions of the
amino acid sequence of the protein or polypeptide to be analyzed,
said peptides being either contiguous, or preferably overlapping
each other, the amount of overlapping being at least 3 amino acids,
and preferably about 6 to about 12, the length of the peptides
being at least about 5 amino acids and no more than about 50,
preferably no more than about 40 amino acids, and more preferably
from 9 to about 30 amino acids, with said peptides being
characterized in that they are biotinylated;
[0147] binding the peptides to a solid phase through the
interaction between the biotinyl group and streptavidin or avidin
and measuring antibody binding to the individual peptides using
classical methods.
[0148] (9) The present invention also relates to a process for the
in vitro determination of antibodies to HIV or diagnosis of HIV
infection by using a peptide composition as defined above in an
immunoassay procedure, wherein the biotinylated peptides used are
in the form of complexes of streptavidin-biotinylated or of:
avidin-biotinylated peptides.
[0149] (10) The present invention relates also to a process for the
in vitro determination of antibodies to HCV or diagnosis of HCV
infection by using a peptide composition as defined above in an
immunoassay procedure, wherein the biotinylated peptides used are
in the form of complexes of streptavidin-biotinylated or of:
avidin-biotinylated peptides.
[0150] (11) The present invention relates also to a process for the
in vitro determination of antibodies to HTLV I or II or diagnosis
of HTLV I or II infection by using a peptide composition as defined
above in an immunoassay procedure, wherein the biotinylated
peptides used are in the form of complexes of
streptavidin-biotinylated or of avidin-biotinylated peptides.
[0151] A preferred method for carrying out the in vitro
determination of antibodies is by means of an enzyme-linked
immunosorbant assay (ELISA). This assay employs a solid phase which
is generally a polystyrene microtiter plate or bead. The solid
phase may, however, be any material which is capable of binding a
protein, either chemically via a covalent linkage or by passive
adsorption. In this regard, nylon-based membranes are also
considered to be particularly advantageous. The solid phase is
coated with streptavidin or avidin and after a suitable period,
excess unbound protein is removed by washing. Any unoccupied
binding sites on the solid phase are then blocked with an
irrelevant protein such as bovine serum albumin or casein.
[0152] A solution containing the mixture or selection of
biotinylated peptides is subsequently brought into contact with the
streptavidin- or avidin-coated surface and allowed to bind. Unbound
peptide is removed by washing. Alternatively biotinylated peptides
are allowed to form complexes with either avidin or streptavidin.
The resulting complexes are used to coat the solid phase. After a
suitable incubation period, unbound complex is removed by washing.
An appropriate dilution of an antiserum or other body fluid is
brought into contact with the solid phase to which the peptide is
bound. The incubation is carried out for a time necessary to allow
the binding reaction to occur. Subsequently, unbound components are
removed by washing the solid phase. The detection of immune
complexes is achieved by using heterologous antibodies which
specifically bind to the antibodies present in the test serum and
which have been conjugated with an enzyme, preferably but not
limited to either horseradish peroxidase, alkaline phosphatase, or
.beta.-galactosidase, which is capable of converting a colorless or
nearly colorless substrate or co-substrate into a highly colored
product or a product capable of forming a colored complex with a
chromogen which can be detected visually or measured
spectrophotometrically.
[0153] Other detection systems known in the art may however be
employed and include those in which the amount of product formed is
measured electrochemically or luminometrically. The detection
system may also employ radioactively labelled antibodies, in which
case the amount of immune complex is quantified by scintillation
counting or counting. In principle, any type of immunological test
for the detection of antibodies may be used, as long as the test
makes use of the complex between either streptavidin or avidin and
(a) biotinylated peptide(s) synthesized as described.
[0154] Also included are competition assays in which streptavidin-
or avidin-biotinylated peptide complexes in solution aria permitted
to compete with the solid phase-bound antigen for antibody binding
or assays in which free peptide in solution is permitted to compete
with solid phase-bound streptavidin or avidin: biotinylated peptide
complexes. By way of example, the many types of immunological
assays for the detection and quantitation of antibodies and antigen
are discussed in detail (Tijssen, P., Practice and Theory of Enzyme
Immunoassays, Elsevier Press, Amsterdam, Oxford, New
[0155] York, 1985).
[0156] The immunological assays may be restricted to single
biotinylated peptides. Preferably, however, a mixture of
biotinylated peptides is used which includes more than one epitope
derived from the infectious agent(s) to which the presence of
specific antibodies is to be measured.
[0157] Another preferred method for carrying out the in vitro
determination of antibody detection is the line immunoassay
(LIA).
[0158] This method of antibody detection consists essentially of
the following steps:
[0159] the antigens, in the form of biotinylated peptide:
streptavidin or avidin complexes, to be tested or used are applied
as parallel lines onto a membrane which is capable of binding,
covalently or non-covalently, the antigen to be tested,
[0160] unoccupied binding sites on the membrane are blocked with an
irrelevant protein such as casein or bovine serum albumin,
[0161] the membrane is cut into strips in a direction perpendicular
to the direction in which the antigen (biotinylated peptide) lines
are applied,
[0162] an appropriate dilution of an antiserum or other body fluid
(containing antibodies to be detected) is brought into contact with
a strip to which the antigens are bound and allowed to incubate for
a period of time sufficient to permit the binding reaction to
occur,
[0163] unbound components are removed by washing the strip,
[0164] the detection of immune complexes is achieved by incubating
the strip with heterologous antibodies which specifically bind to
the antibodies in the test serum and which have been conjugated to
an enzyme such as horseradish peroxidase,
[0165] the incubation is carried out for a period sufficient to
allow binding to occur,
[0166] the presence of bound conjugate is detected by addition of
the required substrate or co-substrates which are converted to a
colored product by the action of the enzyme,
[0167] the reactions are detected visually or may be quantified by
densitometry.
[0168] (12) As demonstrated in the Examples section the present
invention relates also the use of a peptide composition as defined
above, for immunization against HIV, and/or HCV, and/or HTLV I or
II infection.
[0169] (13) The present invention also relates to a method for
preparing the biotinylated peptides used in the invention involves
the use of N-.alpha.-Fmoc-X (N-y-biotin) or N-.alpha.-Fmoc-X
(N-y-biotin) derivative, wherein X represents 1
[0170] where n is at least 1 but less than 10 and is preferably
between 2 and 6, one amino group being attached to the Ca atom
while the other being attached to carbon Cy, which is the most
distal carbon in the side chain; or their esters obtained with
alcohol ROH and more particularly pentafluorophenyl ester;
[0171] y representing position y with respect to the carbon atom
carrying the COOH group in the radical.
[0172] This biotin derivative will be called intermediary product,
and the above-defined intermediary products are new compounds
determined according to the process of the invention.
[0173] (14) In an advantageous method for preparing the compounds
of the invention, the intermediary product can be represented by
one of the following formula:
N-.alpha.-Fmoc-(N-y-biotin) is N-.alpha.-Fmoc-lysine
(.epsilon.-biotin) or N-.alpha.-Fmoc-ornithine
(N-.delta.-biotin)
[0174] (15) The N-terminal biotinylated peptides can be prepared
according to the method which comprises the following steps:
[0175] addition of the successive amino acids duly protected onto
the resin to give:
Fmoc-AA.sub.n . . . AA.sub.1-resin,
[0176] deprotection of the NH.sub.2-terminal for instance by means
of piperidine,
[0177] addition of the intermediary product: 2
[0178] through its COOH onto the NH.sub.2-terminal to obtain: 3
[0179] deprotection of the NH.sub.2-terminal group of the compound
obtained, cleavage from the resin, extraction and purification of
the peptide obtained, biotinylated at its amino terminal, the steps
of side chain deprotection and peptide cleavage being liable to be
performed simultaneously or separately, and particularly
[0180] deprotection of the NH.sub.2-terminal group of the
intermediary group, for instance by means of piperidine,
[0181] cleavage from the resin for instance with an acid such as
trifluoroacetic acid, in the presence of scavengers such as
ethanedithiol, thioanisole, or anisole,
[0182] extraction of the peptide with a solvent such as
diethylether to remove most the acid and scavengers, purification,
such as with HPLC to obtain: 4
[0183] Biotin can be conveniently coupled to the free
amino-terminus (of an otherwise fully protected peptide chain using
also conventional activation procedures. Since biotin possesses one
carboxyl group and no amino groups, biotin essentially functions as
a chain terminator. Preferred activating agents for in situ
activation include but are not limited to
benzotriazol-1-yl-oxy-tripyrrolidinophosphonium hexafluorophosphate
(PyBOP), 0-benzotriazol-1-yl-N,N,N',N'-tetramethyluro- nium
hexafluorophosphate (HBTU), and
O-(1H-benzotriazol-1-yl)-N,N,N',N'-te- tramethyluronium
tetrafluoroborate (TBTU). The activation procedures employing these
and related compounds are known to those versed in the art of solid
phase peptide synthesis and the coupling of biotin does not entail
a significant departure from standard coupling protocols.
[0184] Biotin in a pre-activated form may also be used. Either
N-hydroxysuccinimidobiotin or biotinamidocaproate
N-hydroxysuccinimide ester are conveniently employed and both are
commercially available. This method of coupling has been described
by Lobl, T. J., Deibel, M. R., and Yem, A. W., Anal. Biochem.
(1988) 170(2):502-511. Following addition of the N-terminal biotin,
the peptide is cleaved from the resin in the presence of
scavengers, the choice of which will depend on the usual
considerations of peptide amino acid composition and the nature of
the protecting groups used.
[0185] (16) The carboxy terminal biotinylated peptides involved in
the process of the invention can be prepared according to a method
which comprises
[0186] coupling of a carboxy-activated form of the intermediary
product as defined above to a cleavable linker attached to the
resin, for instance to obtain the following compound: 5
[0187] deprotection of the .alpha. amino group of the intermediary
compound, for instance by means of piperidine to obtain: 6
[0188] sucessive addition of the subsequent amino acids AA.sub.1. .
. AA.sub.n dulu protected onto 7
[0189] to obtain: 8
[0190] deprotection of the NH.sub.2-terminal for instance by means
of piperidine,
[0191] deprotection of the compound obtained, cleavage from the
resin, extraction and purification of the peptide obtained,
biotinylated at its carboxy terminal end, the steps of side chain
deprotection and peptide cleavage being liable to be performed
simultaneously or separately, and particularly
[0192] deprotection of the NH.sub.2-terminal, for instance by means
of piperldine,
[0193] cleavage from the resin for instance with trifluoroacetic
acid, in the presence of scavengers such as ethanedithiol, or
thioanisole, or anisole,
[0194] extraction of the peptide with a solvent such as
diethylether to remove most of the acid and scavengers,
[0195] purification, such as with HPLC to obtain: 9
[0196] (17) The internally biotinylated peptides can be prepared
according to a method which comprises the following steps:
[0197] addition of successive amino acids duly protected onto the
resin to give:
Fmoc-AA.sub.n . . . AA.sub.1-resin,
[0198] deprotection of the NH.sub.2-terminal, --addition of the
intermediary product: 10
[0199] through its COOH onto the NH.sub.2-terminal to obtain:
11
[0200] deprotection of the .alpha. amino group of the intermediary
compound, for instance by means of piperidine to obtain: 12
[0201] addition of the subsequent amino acids duly protected onto
the resin to give: 13
[0202] deprotection of the NH.sub.2 terminal group of the compound
obtained, cleavage from the resin, extraction and purification of
the peptide obtained, biotinylated at its amino-terminal, the steps
of side chain deprotection and peptide cleavage being liable to be
performed simultaneously or separately, and particularly,
[0203] deprotection of the NH.sub.2-terminus, for instance by means
of piperidine,
[0204] cleavage from the resin for instance with trifluoroacetic
acid, in the presence of scavengers such as ethanedithiol, or
thioanisole, or anisole,
[0205] extraction of the peptide with a solvent such as
diethylether to remove most of the acid and scavengers,
purification, such as with HPLC to obtain: 14
[0206] Under certain circumstances, it may prove particularly
advantageous to be able to biotinylate a peptide internally
[0207] or at its carboxy-terminus. Such instances arise, for
example, when the amino acid sequence of a peptide corresponds to
the amino-terminal sequence of a protein.
[0208] Attachment of a biotin to the amino-terminus of such a
peptide results in a structure which is significantly different
from that found in the native protein and may, as a consequence,
adversely affect the binding properties of biochemical properties
of the peptide. It is also possible that even for peptides
corresponding to internal protein sequences, their recognition by
binding proteins or immunoglobulins may depend on which end of the
peptide and then manner in which it is presented for binding. The
importance of peptide orientation has been described by Dyrberg, T.
and Oldstone, M. B. A., J. Exp. Med. (1986) 164:1344-1349.
[0209] In order to be able to incorporate a biotinyl moiety into a
peptide in a position and sequence independent manner, efforts were
made to synthesize a suitable reagent which can be coupled using
conventional procedures. A convenient reagent for C-terminal or
internal biotinylation is N-.epsilon.-biotinyl-lysine. Provided the
a-amino group of this compound is suitably protected (Fmoc and
tBoc), this reagent may be used to introduce a biotin anywhere in
the peptide chain, including at the amino terminus, by the standard
procedures used in solid phase peptide synthesis.
[0210] The synthesis of the t-Boc-protected derivative has been
described (Bodansky, M., and Fagan, D T., J. Am. Chem. Soc. (1977)
99:235-239) and was used to synthesize short peptides for use in
studying the enzyme activities of certain transcarboxylases.
[0211] Unlike the t-Boc derivative, the synthesis of
N-.alpha.-Fmoc-Lys (N-.epsilon.-biotin) has not been described and
given the growing interest in Fmoc-based synthesis strategies, this
compound is considered particularly advantageous.
[0212] There are a number of possible routes which can be taken to
arrive at the desired Fmoc-protected compound.
[0213] These are shown in FIG. 1. In the first approach,
commercially available N-.alpha.-Fmoc-Lys (N-.epsilon.-tBoc) can be
used as the starting material. The N-.epsilon.-tBoc protection is
removed using trifluoroacetic acid and a scavenger such as water. A
slight molar excess of the N-.epsilon.-Fmoc-lysine so obtained is
then reacted with carboxy-activated biotin. The resulting product
can be readily purified by selective extractions and standard
chromatographic techniques. In an alternative approach,
N-.alpha.-Fmoc-Lys (N-.epsilon.-biotin) can be produced from
commercially available N-.epsilon.-biotinyl lysine (biocytin) by
reaction with fluorenylmethylsuccinimidyl carbonate. Numerous
examples of these reactions which can be used as guidelines are
given in Atherton and Sheppard, Solid Phase Peptide Synthesis, IRL
Press, 1989.
[0214] The strategy shown in FIG. 1 (method A) may also be applied
to synthesize N-.alpha.-Fmoc-ornithine (N-.delta.-biotin) from
commercially available N-.alpha.-Fmoc-ornithine (N-.delta.-tBoc).
The ornithine derivative differs from the lysine derivative only in
the length of the side chain which, for the ornithine derivative,
is shorter by one carbon atom.
[0215] The N-.alpha.-Fmoc-Lys can be conveniently incorporated into
the peptide chain using the same reagents for in situ activation
described for free biotin.
[0216] Alternatively, N-.alpha.-Fmoc-Lys
(N-.epsilon.-biotin)-O-pentafluor- ophenyl ester can be
conveniently synthesized from N-.alpha.-Fmoc-Lys
(N-.epsilon.-biotin) and pentafluorophenyl trifluoroacetate using
the base-catalyzed transesterification reaction described by Green,
1K. and Berman, J., Tetrahedron Lett. (1990) 31:5851-5852, for the
preparation of O-pentafluorophenyl esters of amino acids.
[0217] This active ester can be used directly to incorporate
N-.alpha.-Fmoc-Lys (N-.delta.-biotin) into the peptide chain. The
class of above-defined intermediary products can be prepared
according to a method which comprises the following steps:
[0218] reaction of a diamino-, monocarboxylic acid previously
described with fluorenylmethysuccinimidylcarbonate or
fluorenylmethyl chloroformate under conditions of carefully
controlled pH to give the singly protected N-.alpha.-Fmoc
derivative,
[0219] or alternatively, use of commercially available
N-.alpha.-Fmoc-protected diamino-monocarboxylic acids when the side
chain amino group is provided with a protecting group which is
different from the Fmoc group used to protect the .alpha.-amino
group, the side chain amino group protection being liable to be
selectively removed under conditions which leave the N-.alpha.-Fmoc
group intact,
[0220] purification of the mono-protected
N-.alpha.-Fmoc-diamino-monocarbo- xylic acid derivative by
selective extractions and chromatography,
[0221] reaction of the derivative obtained with a carboxy-activated
derivative of biotin, such as N-hydroxysuccinimide biotin, to
obtain the (N-.alpha.-Fmoc)-(N-y-biotin) derivative which is the
desired intermediary product,
[0222] purification of the intermediary product by selective
extractions, precipitations, or chromatography.
[0223] When the biotinylated peptides used in the process of the
invention are to be provided with linker arms, these chemical
entities may be conveniently attached- to either the N- or
C-terminus of a peptide sequence during solid phase synthesis using
standard coupling protocols, as long as the amino groups of these
compounds are provided with appropriate temporary amino group
protection.
[0224] All these specific biotinylated peptides are new.
DESCRIPTION OF THE FIGURES
[0225] All the samples and sera mentioned in the figures and tables
are randomly chosen samples and sera, containing antibodies
produced as a result of naturally occurring infection by a viral
agent.
[0226] FIGS. 1a-1c represent the strategies for the synthesis of
N-.alpha.-Fmoc-lysine (N-.epsilon.-Biotin)
[0227] More particularly:
[0228] Method A corresponds to the synthesis of (N-.alpha.-Fmoc-Lys
(N-.epsilon.-biotin) from N-.epsilon.-Fmoc-Lys (N-.epsilon.-tBoc)
and Method B corresponds to the synthesis of (N-.alpha.-Fmoc-Lys
(N-.epsilon.-biotin) from N-.epsilon.-biotinyl lysine.
[0229] FIGS. 2a and 2b represent the diagram obtained in reverse
phase chromatography of the precursors involved in the preparation
of the intermediary products defined above, and of the intermediary
compounds.
[0230] The reverse phase chromatography has been carried out in the
following conditions:
[0231] gradient specifications:
[0232] buffer A: 0.1% TFA in H20,
[0233] buffer B: 0.1% TFA in acetonitrile,
[0234] column: C2/C18 reverse phase (Pharmacia, Pep-S),
[0235] detection wavelength: 255 nanometers;
[0236] gradient:
[0237] 0% B from 0 to 1 minute,
[0238] 0% B to 100% B from 1 minute to 60 minutes,
[0239] 0% B from 60 minutes to 70 minutes.
[0240] The first diagram corresponds to method A (see FIG. 1) and
the; second diagram corresponds to method B (see FIG. 1).
[0241] FIGS. 3a-1 and 3a-2 represent the antibody binding to HCV
peptide II (in an ELISA).
[0242] The upper left curve corresponds to sample 8320.
[0243] The upper right curve corresponds to sample 8242.
[0244] The lower left curve corresponds to sample 8243.
[0245] The lower right curve corresponds to sample 8318.
[0246] In each of these samples, the optical density (at 450 nm) is
plotted against the coating concentration expressed in
.mu.g/ml.
[0247] The curve with crosses corresponds to non-biotinylated HCV
peptide II and the curve with dots corresponds to biotinylated HCV
peptide II.
[0248] FIGS. 3b-1 and 3b-2 represent the antibody binding to HCV
peptide XI (in an ELISA).
[0249] The upper left curve corresponds to sample 8320.
[0250] The upper right curve corresponds to sample 8326.
[0251] The lower left curve corresponds to sample 8242.
[0252] The lower right curve corresponds to sample 8243.
[0253] In each of these samples, the optical density (at 450 nm) is
plotted against the coating concentration expressed in
.mu.g/ml.
[0254] The curve with crosses corresponds to non-biotinylated HCV
peptide XI and the curve with dots corresponds to biotinylated HCV
peptide XI.
[0255] FIGS. 3c-1 and 3c-2 represent the antibody binding to HCV
peptide XVI (in an ELISA).
[0256] The upper left curve corresponds to sample 8326.
[0257] The upper right curve corresponds to sample 8242.
[0258] The lower left curve corresponds to sample 8243.
[0259] The lower right curve corresponds to sample 8318.
[0260] In each of these samples, the optical density (at 450 nm) is
plotted against the coating concentration expressed in
.mu.g/ml.
[0261] The curve with crosses corresponds to non-biotinylated HCV
peptide XVI and the curve with dots corresponds to biotinylated HCV
peptide XVI.
[0262] FIGS. 4a and 4b correspond to the detection of biotinylated
peptides coated directly (in an ELISA).
[0263] The first curve corresponds to biotinylated HCV peptide II,
the second curve to biotinylated HCV peptide XI and the third curve
to biotinylated HCV peptide XVI.
[0264] In each of these samples, the optical density (at 450 nm) is
plotted against the coating concentration expressed in
.mu.g/ml.
[0265] FIGS. 5a and b represent the structures of N- and
C-terminally biotinylated HIV-1 peptides (hereabove designated by
1a.1) originating from the transmembrane (TM) protein of HIV-1.
[0266] FIGS. 6a-1 through 6a-5 represent the detection of core
epitopes in the Core region of HCV using overlapping 9-mers (in an
ELISA).
[0267] The sera used are indicated above each diagram. The
ordinates correspond to the optical density at 450 nm.
[0268] The abscissae correspond to the sequence of the protein in
which the location of the epitope (s) is to be determined. For
purposes of graphic illustration, the optical density is assigned
to the first amino acid in the respective nine-mer sequences.
[0269] FIGS. 6b-1 through 6b-5 represent the detection of core
epitopes in the NS4 region of HCV using overlapping 9-mers (in an
ELISA).
[0270] The sera used are indicated above each diagram. The
ordinates correspond to the optical density at 450 nm.
[0271] The abscissae correspond to the sequence of the protein in
which the location of the epitope (s) is to be determined. For
purposes of graphic illustration, the optical density is assigned
to the first amino acid in the respective nine-mer sequences.
[0272] FIGS. 6c-1 through 6c-10 represent the detection of core
epitopes in the NS5 region of HCV using overlapping 9-mers (in an
ELISA).
[0273] The sera used are indicated above each diagram. The
ordinates correspond to the optical density at 450 nm.
[0274] The abscissae correspond to the sequence of the protein in
which the location of the epitope (s) is to be determined. For
purposes of graphic illustration, the optical density is assigned
to the first amino acid in the respective nine-mer sequences.
[0275] FIGS. 7a-1 through 7a-3 correspond to the positions of
biotinylated 20-mers with respect to overlapping 9-mers (in an
ELISA).
[0276] The abscissae corresponds to the protein sequence in which
the epitope(s) is to be determined.
[0277] FIGS. 7b-1 through 7b-3 correspond to the positions of
biotinylated 20-mers with respect to overlapping 9-mers (in an
ELISA).
[0278] The abscissae corresponds to the protein sequence in which
the epitope(s) is to be determined.
[0279] FIGS. 7c-1 through 7c-4 correspond to the positions of
biotinylated 20-mers with respect to overlapping 9-mers (in an
ELISA).
[0280] The abscissae corresponds to the protein sequence in which
the epitope(s) is to be determined.
[0281] FIG. 8 represents a comparison of antibody recognition of
biotinylated and unbiotinylated HCV peptides by line immunoassay
(LIA).
[0282] FIG. 9 represents a comparison of antibody recognition of
biotinylated core peptides by line immunoassay (LIA).
[0283] The shorter and longer peptides are compared.
[0284] FIG. 10 represents an evaluation of type-specific HCV NS4
peptides by Line immunoassay (LIA).
[0285] FIG. 11 represents the amino acid sequence of peptides NS4-a
to NS4-e.
[0286] FIG. 12 represents the composition of hybrid HCV
peptides.
[0287] FIG. 13 represents the antibody recognition of hybrid HCV
peptides.
[0288] FIGS. 14a-14d represent the construction scheme for mixotope
peptides from the N-terminus of E2/NS1 of HCV type 1.
[0289] FIG. 15 represents the mixotope synthesis strategy.
[0290] FIGS. 16A and 16B represent the synthesis of multiple
antigen peptides (MAPs).
[0291] FIGS. 17A and 17B represent the recognition of E2/NS1
peptides, by sera from rabbits immunized with E2/NS1 "b" peptide
MAPs.
[0292] FIG. 18 represents the recognition of a commercially
available serum panel with a number of biotinylated HTLV-I and
HTLV-II peptides incorporated into LIA strips.
[0293] Table 1 represents the antibody recognition of
unbiotinylated HIV-1 and HIV-2 peptides (designated by TM-HIV1 and
TM-HIV-2) and biotinylated HIV-1 and HIV-2 peptides (hereabove
referred to as 1a.1 and 2a, and also designated by TM-HIV-1 Bio and
TM-HIV-2 Bio) in an ELISA.
[0294] Table 2 represents the comparison of antibody recognition of
unbiotinylated and biotinylated peptides from the V3 sequence of
isolate HIV-1 nm (also referred as 1b.4) in an ELISA.
[0295] Table 3 represents the comparison of antibody recognition of
the biotinylated V3"mn peptide (referred to as 1b.4) bound to
streptavidin and avidin, in an ELISA.
[0296] Table 4 represents the comparison of antibody recognition of
biotinylated and unbiotinylated HCV peptides, in an ELISA.
[0297] More particularly:
[0298] Table 4A corresponds to the antibody binding to HCV peptide
XI.
[0299] Table 4B corresponds to the antibody binding to HCV peptide
XVI.
[0300] Table 4C corresponds to the antibody binding to HCV peptide
II.
[0301] Table 4D corresponds to the antibody binding to HCV peptide
III.
[0302] Table 4E corresponds to the antibody binding to HCV peptide
V.
[0303] Table 4F corresponds to the antibody binding to HCV peptide
IX.
[0304] Table 4G corresponds to the antibody binding to HCV peptide
XVIII.
[0305] Table 5 represents a comparison of antibody binding to
biotinylated and non-biotinylated peptides, at different peptide
coating concentrations, in an ELISA.
[0306] Table 6 represents the comparison of N- and C-terminally
biotinylated TM-HIV-1 peptide (referred to as 1a.1), in an
ELISA.
[0307] Table 7 represents a comparison of antibody recognition of
unbiotinylated and carboxy-biotinylated HCV peptide I.
[0308] Table 8 represents the use of mixtures of biotinylated HIV
and HCV peptides for antibody detection, in an ELISA.
[0309] Table 9 represents sequences of the core epitopes of the HCV
Core protein.
[0310] Table 10 represents sequences of the core epitopes of the
HCV NS4 protein.
[0311] Table 11 represents sequences of the core epitopes of the
HCV NS5 protein.
[0312] Table 12 represents the antibody binding of various Core,
NS4, and NS5 biotinylated 20-mers by 10 test sera.
[0313] Table 13 represents the antibody recognition of individual
E2/NS1 peptides (percent of all sera giving a positive
reaction).
[0314] Table 14 represents the overall recognition of HIV V3-loop
peptides.
[0315] Table 15 represents the recognition of HIV peptides
according to the geographical region.
[0316] Table 16 represents the recognition of European, African and
Brazilian HIV-1-positive sera to HIV-I V3-loop peptides V3-con and
V3-368.
[0317] Table 17 represents the recognition of HIV-2 positive sera
to two HIV-2 V3 loop peptides.
[0318] Table 18 represents the antibody recognition of hybrid
peptides.
[0319] Table 19 represents the antibody recognition of mixed HTLVI
and II peptides.
[0320] All amino acid sequences are given in the conventional and
universally accepted three-letter code and where indicated in the
one-letter code. The peptide sequences are given left to right
which, by convention, is the direction from the amino terminus to
the carboxy-terminus.
[0321] A number of unconventional codes are also used to represent
chemical groups or modifications and are defined as follows:
33 Group Code Ac acetyl Bio D-biotinyl Fmoc
9-fluorenylmethoxycarbonyl tBoc tertiary butyloxycarbonyl
EXAMPLE 1
Peptide Synthesis
[0322] All of the peptides described were synthesized on TentaGel
S-RAM (Rapp Polymere, Tubingen, Germany), a
polystyrene-polyoxyethylene graft copolymer functionalized with the
acid-labile linker
4-(.alpha.-Fmoc-amino-2',4'-dimethoxybenzyl)phenoxyacetic acid
(Rink, Tetrahedron Lett. (1987) 28:3787) in order to generate
peptide carboxy-terminal amides upon cleavage. t-Butyl-based side
chain protection and Fmoc-.alpha.-amino-protection was used.
[0323] The guanidine-group of arginine was protected with the
2,2,5,7,8-pentamethylchroman-6-sulfonyl moiety. The imidazole group
of histidine was protected with either t-Boc or trityl and the
sulfhydryl group of cysteine was protected with a trityl group.
Couplings were carried out using preformed O-pentafluorophenyl
esters except in the case of arginine where TBTU was used as the
activating agent in the presence of 1.5 equivalents of the base
N-methylmorpholine. Occasionally, glutamine and asparagine were
also coupled using TBTU activation. In these cases, the
trityl-protected derivatives of these amino acids were employed.
Biotin was coupled using either TBTU or HBTU. All syntheses were
carried out on a Milligen 9050 PepSynthesizer (Novato, Calif.)
using continuous flow procedures. Following cleavage with
trifluoroacetic acid in the presence of scavengers and extraction
with diethylether, all peptides were analyzed by C18-reverse phase
chromatography.
EXAMPLE 2
Synthesis of N-.alpha.-Fmoc-Lys (N-.epsilon.-biotin)
[0324] A. Method A
[0325] Commercially available N-.alpha.-Fmoc-L-lysine
(N-.epsilon.-tBoc) (1.5 grams) was treated with 20 milliliters of
95% trifluoroacetic acid, 5% H.sub.2O for 2 hours at room
temperature. Most of the acid was then evaporated under a stream of
nitrogen. Ten milliliters of water was added and the solution was
extracted 3 times with diethylether. The aqueous phase was then
evaporated to dryness in vacuo over phosphorus pentoxide. The
resulting powder (N-.alpha.-Fmoc-L-lysine) was analyzed by reverse
phase chromatography and revealed a homogeneous product which was,
as expected, more hydrophilic than the starting material.
[0326] N-.alpha.-Fmoc-lysine (190 mg, 0.49 mmol) was dissolved in 8
milliliters of 0.1 M borate buffer, pH 8.7.
N-hydroxysuccinimidobiotin (162 mg, 0.47 mmol) was dissolved in 4
milliliters of dimethylformamide and added to the solution of
N-.alpha.-Fmoc-lysine. The pH was monitored and titrated as
necessary, with NaOH. After 2 hours, the solution was acidified
with HCl to pH 2.0, at which time a white precipitate was
obtained.
[0327] Following extraction with ethylacetate and centrifugation,
the white precipitate was found at the H2O: ethylacetate interface.
Both phases were removed and the precipitate extracted twice with
10 mM HCl, once with ethylacetate, followed by two extractions
[0328] with diethylether. The precipitate was dissolved in DMF and
precipitated by addition of diethylether. The crystalline powder
was then dried in vacuo over phosphorus pentoxide. The resulting
product was analyzed by reverse phase chromatography and revealed a
major peak which, as expected, eluted later than
N-.alpha.-Fmoc-Lys. A very small peak of N-.alpha.-Fmoc-Lys was
also observed. (FIG. 2a)
[0329] B. Method B
[0330] Commercially available N-.epsilon.-biotinyl lysine
(biocytin, Sigma, 249 mg, 0.67 mmol) was dissolved in 8 milliliters
of 1 M Na.sub.2CO.sub.3 and cooled on ice.
Fluorenylmethylsuccinimidyl carbonate (222 mg, 0.66 mmol) was
dissolved in 2 milliliters of acetone and was added to the biotinyl
lysine solution over a period of 30 minutes with vigorous stirring.
Stirring was continued for 5 hours at room temperature. The pH was
maintained between 8 and 9 by addition of 1 M Na.sub.2CO.sub.3 as
necessary. The acetone was then evaporated off under vacuum, and
1.0 M HCl was added until the pH of the solution was approximately
2. Upon acidification of the solution, a white precipitate appeared
which was washed twice with 10 mM HCl, twice with ethyl acetate,
and twice with diethylether. The precipitate was dissolved in DMF
and precipitated by addition of diethylether. The crystalline
powder was then thoroughly dried in vacuum over phosphorus
pentoxide. The resulting product was analyzed by reverse phase
chromatography and revealed a major peak which eluted with the same
retention time (30.5 minutes) as the product obtained using method
1 (FIG. 2b).
EXAMPLE 3
Methods for the Determination of Peptides Corresponding to
Immunologically Important Epitopes in an Enzyme-Linked
Immunosorbent Assay (Elisa) Using Specific Antibodies
[0331] Where peptides were to be coated directly, stock solutions
of the peptides were diluted in sodium carbonate buffer, pH 9.6 and
used to coat polystyrene microtiter plates at a peptide
concentration of 2 to 5 micrograms per milliliter for 1 hour at
37.degree. C.
[0332] In cases where biotinylated peptides were to be evaluated,
plates were first coated with streptavidin in sodium carbonate
buffer, pH 9.6 at a concentration of 3 micrograms per milliliter
for 1 hour at 37.degree. C. The plates were then washed to remove
excess, unbound protein. A working solution of the biotinylated
peptide at 1 microgram per milliliter in sodium carbonate buffer
was then added to the wells of the microtiter plate and incubated
for 1 hours at 37.degree. C.
[0333] Once the plates had been coated with antigen, any remaining
free binding sites on the plastic were blocked with casein. After
washing, a dilution of the appropriate antisera, usually 1:100, was
added to the wells of the plates and incubated for 1 hour at
37.degree. C. After washing to remove unbound material, specific
antibody binding was detected by incubating the plates with goat
anti-human immunoglobulin antibodies conjugated to the enzyme
horseradish peroxidase.
[0334] Following removal of unbound conjugate by washing, a
solution containing H.sub.2O.sub.2 and
3,3',5,5'-tetramethylbenzidine was added.
[0335] Reactions were stopped after a suitable interval by addition
of sulfuric acid. Positive reactions gave rise to a yellow color
which was quantified using a conventional microtiter plate reader.
Absorbance measurements were made at a wavelength of 450 nanometers
and all data are expressed as an optical density value at this
wavelength.
EXAMPLE 4
Use of Biotinylated HIV Peptides for the Detection of HIV-Specific
Antibodies
[0336] Experiments were performed to evaluate antibody recognition
of short, 10 amino acid-long, N-acetylated peptides corresponding
to other contained within the transmembrane proteins of HIV-1 and
HIV-2. Direct coating of these peptides in the wells of microtiter
plates gave very poor results when antibody binding was evaluated
in an ELISA. Since it was suspected that the peptides did not bind
well to the polystyrene solid phase, the peptides were
resynthesized in the same way except that biotin was attached to
the amino terminus of the peptides, separated from the decamer
peptide sequence by three glycine residues whose function it was to
serve as a linker arm. The peptides used for the comparison were as
follows:
34 TM-HIV-1: Ac-Ile-Trp-Gly-Cys-Ser-Gly-Lys- (SEQ ID NO:110)
Leu-Ile-Cys-NH.sub.2 TM-HIV-1 Bio Bio-Gly-Gly-Gly-Ile-Trp-Gly-Cys-
(SEQ ID NO:111) Ser-Gly-Lys-Leu-Ile-Cys-NH.sub.2 TM-HIV-2
Ac-Ser-Trp-Gly-Cys-Ala-Phe-Arg- (SEQ ID NO:112)
Gln-Val-Cys-NH.sub.2 TM-HIV-2 Bio Bio-Gly-Gly-Gly-Ser-Trp-Gly-Cys-
(SEQ ID NO:113) Ala-Phe-Arg-Gln-Val-Cys-NH.sub.2
[0337] The biotinylated peptides were loaded onto microtiter plates
which had been coated with streptavidin. Antibody binding to these
peptides was compared to antibody binding to the unbiotinylated
peptides which were coated directly onto microtiter plates. The
results are shown in Table 1. It is evident that the biotinylated
peptides from the HIV-1 or HIV-2 transmembrane proteins bound to
streptavidin are recognized very well by antisera from HIV-1 or
HIV-2 infected persons respectively. This is in contrast to the
unbiotinylated versions of these peptides coated directly onto the
polystyrene plates. Addition control experiments showed that the
increase in antibody binding was the result of the specific
interaction between the biotinylated peptide and streptavidin,
since there was no difference in antibody recognition of the
biotinylated or unbiotinylated peptides when both were coated
directly onto the microtiter plate.
[0338] Some peptides, particularly ones which are 15 amino acids in
length or longer, bind sufficiently to the solid phase to allow the
detection of specific antibodies which recognize (an) epitope(s)
present in the peptide sequence.
[0339] To ascertain whether biotinylation would also improve
antibody recognition of longer peptides, both the biotinylated and
unbiotinylated versions of the partial V3 loop sequence of isolate
HIV-1 nm were synthesized. The sequence and method of synthesis of
both peptides were identical except at the amino terminus. The
unbiotinylated peptide was simply acetylated whereas in the
biotinylated version, two glycine residues were added as a linker
arm to separate the peptide from the biotinyl moiety.
[0340] The sequences of the two peptides used are as follows:
35 unbiotinylated V3 mn peptide Ac-Tyr-Asn-Lys-Arg-Lys-Arg- -Ile-
(SEQ ID NO:114) His-Ile-Gly-Pro-Gly-Arg-Ala-Phe-
Tyr-Thr-Thr-Lys-Asn-Ile-Ile-Gly- NH.sub.2, biotinylated V3 mn
peptide (peptide 1b.4) Bio-Gly-Gly-Tyr-Asn-Lys-Arg-Lys- (SEQ ID
NO:115) Arg-Ile-His-Ile-Gly-Pro-Gly-Arg- Ala-Phe-Tyr-Thr-Thr-Lys--
Asn-Ile- Ile-Gly-NH.sub.2.
[0341] The unbiotinylated peptide was coated directly onto the
wells of a polystyrene microtiter plate while the biotinylated
peptide was bound to wells which had previously been coated with
streptavidin. The results shown in Table 2 demonstrate that
antibody binding to the biotinylated peptide is superior to
antibody binding to peptide coated directly onto the plastic.
EXAMPLE 5
Use of Biotinylated Peptides-Avidin Complexes for Antibody
Detection
[0342] Having demonstrated that antibody recognition of this
peptide is improved when the peptide is biotinylated and bound to
streptavidin, an additional experiment was performed to determine
whether streptavidin could be substituted by avidin. The results
shown in Table 3 indicate that this is the case and that
biotinylated peptides bound to avidin are recognized very
efficiently by specific antibodies.
EXAMPLE 6
Use of Biotinylated HCV Peptides for Detection of HCV Specific
Antibodies
[0343] In order to determine whether the enhanced antibody
recognition of biotinylated peptides was a general phenomenon, a
number of additional twenty amino acid-long peptides were
synthesized which correspond to sequences derived from the
hepatitis C virus (HCV) polyprotein. The amino acid sequences
evaluated were as follows:
36 a. HCV peptide XI Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu- (SEQ ID
NO:116) Gln-Gly-Met-Met-Leu-Ala-Glu-Gln- Phe-Lys-Gln-Lys b. HCV
peptide XVI Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala- (SEQ ID NO:117)
Gln-Ala-Leu-Pro-Val-Trp-Ala-Arg- Pro-Asp-Tyr-Asn c. HCV peptide II
Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn- (SEQ ID NO:118)
Thr-Asn-Arg-Arg-Pro-Gln-Asp-Val- Lys-Phe-Pro-Gly d. HCV peptide III
Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln- (SEQ ID NO:119)
Asp-Val-Lys-Phe-Pro-Gly-Gly-Gly- Gln-Ile-Val-Gly e. HCV peptide V
Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser- (SEQ ID NO:120)
Gln-Pro-Arg-Gly-Arg-Arg-Gln-Pro- Ile-Pro-Lys-Val f. HCV peptide IX
Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu- (SEQ ID NO:121)
Tyr-Arg-Glu-Phe-Asp-Glu-Met-Glu- Glu-Cys-Ser-Gln g. HCV peptide
XVIII Glu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr- (SEQ ID NO:122)
Glu-Pro-Pro-Val-Val-His-Gly-Cys- Pro-Leu-Pro-Pro
[0344] In each case, two versions of the peptide were synthesized.
In the unbiotinylated version, the peptide was acetylated at the
amino terminus. The biotinylated versions were all N-terminally
biotinylated. A linker arm consisting of two glycine residues
separated the biotinyl moiety from the amino acids comprising the
HCV sequence.
[0345] The unbiotinylated peptides were adsorbed onto the wells of
polystyrene microtiter plates at a concentration of 3 micrograms
per milliliter.
[0346] The biotinylated peptides were bound at a concentration of 1
microgram per milliliter to streptavidin-coated microtiter plates.
Sera known to contain antibodies to these peptides were used for
the evaluation and were tested at a 20-fold dilution. The results
of these comparisons are shown in Table 4, a to g. These results
clearly indicate that antibody recognition of biotinylated peptides
bound to streptavidin is enhanced relative to that of peptides
coated directly onto the wells of the microtiter plate.
EXAMPLE 7
Influence of Coating Concentration on Antibody Detection
[0347] To investigate further the enhanced antibody recognition of
biotinylated HCV peptides bound to streptavidin or avidin as
compared to direct adsorption on plastic, the influence of peptide
coating concentration was investigated. Three peptides (HCV
peptides II, XI, and XVI) were coated in concentrations ranging
from 10 nanograms per milliliter to 3 micrograms per milliliter in
a volume of 200 microliters per microtiter plate well. For direct
coating, the unbiotinylated versions of these peptides were used.
The biotinylated versions of these peptides were used to coat wells
to which streptavidin had previously been adsorbed. Sera known to
contain antibodies to these peptides were used at a dilution of 1
to 100 to evaluate the magnitude of antibody binding.
[0348] The numerical results of this experiment are shown in Table
5 and are depicted graphically in FIG. 3a-c.
[0349] It is evident that with few exceptions, the biotinylated
peptide is recognized very well even at the lowest concentration
tested (10 nanograms per milliliter, 2 nanograms per well). In many
cases, optical density values close to the maximum attainable are
observed at a peptide concentration of only 30 nanograms per
milliliter (6 nanograms per well). In contrast, however, the
unbiotinylated peptides adsorbed directly onto the plastic are
poorly bound by antibody, if at all.
EXAMPLE 8
Influence of Biotinylation of Peptides on Coating Efficiency of the
Peptides on a Solid Phase
[0350] To determine if the absence of a signal was due to lack of
peptide adsorption when the peptides were coated directly, an
additional experiment was performed. In this case, the biotinylated
versions of the peptides were coated directly onto the plastic at
the same concentrations used in the previous experiment for the
unbiotinylated versions. To ascertain whether biotin-labeled
peptide was bound, the microtiter plates were incubated with a
streptavidin: horseradish peroxidase conjugate. Since each peptide
contains a single biotinyl group, the resulting optical densities
are a measure of the amount of peptide bound, although the absolute
amount of bound peptide is not known. The results presented
graphically in FIG. 4 demonstrate that plastic-bound peptide can be
detected. As expected, the curves are different for each peptide
which is a reflection of their chemical uniqueness. Two of the
peptides, HCV peptides XI and XVI, appear to bind only weakly to
the wells of the polystyrene microtiter plate and this poor binding
is reflected in the low optical density values obtained in the
ELISA. Since the binding of the biotinylated peptides to
streptavidin-coated wells results in very good antibody
recognition, it is obvious that poor binding of the peptide to the
solid phase is not a limitation when use is made of interaction
between biotin and streptavidin. On the other hand, one of the
peptides, HCV peptide II, shows very significant binding to the
solid phase, particularly at higher coating concentrations.
[0351] However, at no coating concentration did the signal obtained
when the peptide was coated directly ever equal the signal obtained
when the biotinylated peptide was bound to streptavidin. Since even
at the lowest concentration tested, the streptavidin-bound
biotinylated versions of this peptide clearly gives a positive
signal with the antisera tested, the results would seem to indicate
either that the direct coating of this peptide is extraordinarily
inefficient or that other factors are important besides the simple
binding of peptide to the solid phase. Although difficult to
quantify, one of the factors almost certainly involves the manner
in which the peptide is bound and available for antibody binding.
In the case of peptides coated directly onto the solid phase, it is
virtually inevitable that some proportion of the peptide molecules
will interact with the solid phase through amino acid side chains
which are also essential for antibody recognition. These peptide
molecules will therefore be unable to participate in the binding
reaction with antibodies. This problem is not encountered with the
biotinylated peptides which are all bound to the solid phase
through the interaction between biotin and the solid phase-bound
streptavidin.
EXAMPLE 9
Use of C-Terminally Biotinylated HIV Peptides for Specific Antibody
Recognition
[0352] In order to determine whether the peptides biotinylated at
their carboxy-terminus also give use to enhanced antibody
recognition, a carboxy-biotinylated version of the TM-HIV-1 peptide
was synthesized. N-.alpha.-Fmoc-Lys (N-.epsilon.-biotin) prepared
by method A as described was coupled directly to resin
functionalized with the acid labile linker
4-(.alpha.-Fmoc-amino-21,41-dimethoxybenzyl)phenoxyacetic acid
after removal of the linker-bound Fmoc group with 20 percent
piperidine. The coupling was performed using a 3-fold molar excess
of N-.alpha.-Fmoc-Lys (N-.epsilon.-biotin) relative to resin
functional groups. Carboxyl group activation was achieved using one
equivalent of HBTU, one equivalent of 1-hydroxybenzotriazole and
1.5 equivalents of N-methylmorpholine. N-methyl morpholine was
dispensed as a 0.6M solution in dimethylformamide containing 40
percent dimethylsulfoxide which was necessary to achieve complete
dissolution of the N-.alpha.-Fmoc-Lys. (N-.epsilon.-biotin).
Inspection of the Fmoc deprotection peak following coupling of the
N-.alpha.-Fmoc-Lys (N-.epsilon.-biotin) indicated that coupling had
proceeded smoothly and efficiently. Two glycine residues were
coupled to separate the biotinyl lysine from the TM-HIV-1 amino
acid sequence. Following synthesis of the peptide, the amino
terminus was acetylated with acetic anhydride. The resulting
structure of the carboxy-biotinylated peptide differs significantly
from the peptide biotinylated at the amino terminus. A comparison
of these structures is shown in FIG. 5.
[0353] In order to evaluate antibody recognition of these two
peptides, the peptides were bound individually to
streptavidin-coated microtiter plates and tested using a panel of
antisera from HIV-1 seropositive donors. The results of this
comparison is shown in Table 6. Clearly, antibody recognition of
the C-terminally biotinylated peptide compares very favorably with
that of the N-terminally biotinylated peptide. These results also
confirm the utility of the reagent N-.alpha.-Fmoc-Lys
(N-.epsilon.-biotin) for carboxy-terminal biotinylation.
EXAMPLE 10
Comparison of Antibody Recognition of HCV Peptide I, Coated
Directly (Unbiotinylated) or Bound to Streptavidin-Coated Plated
(Carboxy-Terminal Biotinylation)
[0354] A similar experiment was performed using a peptide which
binds relatively well to polystyrene ELISA plates in order to
determine whether the carboxy-biotinylated form of the peptide
would result in superior antibody recognition relative to the
unbiotinylated form of the peptide. The peptide chosen was HCV
peptide I, which was synthesized in the following versions:
[0355] a. unbiotinylated version:
37 H.sub.2N-Met-Ser-Thr-Ile-Pro-Lys-Pro- (SEQ ID NO:123)
Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr- Asn-Arg-Arg-Pro-Gln-CONH.sub.2
[0356] b. carboxy-biotinylated version:
38 H.sub.2N-Met-Ser-Thr-Ile-Pro-Lys-Pro- (SEQ ID NO:124)
Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr- Asn-Arg-Arg-Pro-Gln-Gly-Gly-Lys
(Bio)-CONH.sub.2.
[0357] A spacer consisting of two glycine residues was added at the
carboxy-terminus to physically separate the HCV portion of the
peptide proper from the Lys(N-.epsilon.-Bio) Synthesis was
performed on resin functionalized with
4-(.alpha.-Fmoc-amino-2',4'-dimethoxybenzyl)phenoxyac- etic acid
linker in order to generate carboxy-terminal amides upon cleavage.
Coupling of the N-.alpha.-Fmoc-Lys(N-.epsilon.-biotin) to the
linker was performed using a 3-fold molar excess of the
intermediate product relative to the linker. Activation of the
N-.alpha.-Fmoc-Lys(N-.e- psilon.-biotin) was achieved using one
equivalent of TBTU, one equivalent of 1-hydroxybenzotriazole, and
1.5 equivalents of N-methylmorpholine. The coupling of all other
amino acids was performed according to conventional protocols.
Following cleavage of the peptides in trifluoroacetic acid in the
presence of the appropriate scavengers, the peptides were
precipitated and extracted with diethylether.
[0358] Unbiotinylated HCV peptide I was coated directly onto the
wells of a polystyrene ELISA plate at a concentration of 3
micrograms per milliliter in sodium carbonate buffer, pH 9.6.
Biotinylated HCV peptide I was bound to streptavidin-coated wells
using a stock solution containing the peptide at a concentration of
1 microgram per milliliter. The resulting plates were then
incubated in parallel with a panel of sera from HCV-seropositive
donors. The results of this comparison are shown in Table 7. The
biotinylated peptide clearly gives superior results relative to the
unbiotinylated version of the same sequence. Two of the sera (8326
and 8244) recognize the biotinylated version of this peptide far
better than the unbiotinylated version. The specificity of the
antibody reaction is also reflected by the low optical density
values obtained for 5 serum samples from uninfected donors (F88,
F89, F76, F136, and F6)
EXAMPLE 11
Use of Mixtures of Biotinylated HIV and HCV Peptides
[0359] In many cases, the use of mixtures of peptides is required
to give the desired result. Mixtures of peptides may be used for
the detection of antibodies directed against one or more proteins
of a single virus, or for the detection of antibodies directed
against proteins of several viruses in a single, test. Such tests
are considered particularly advantageous for the screening of blood
donations for their suitability for use in transfusions and as a
source of blood products. In such cases, ELISA plates or other
solid supports coated with suitable mixtures of peptides may be
used to screen samples for the presence of antibodies to one or
more infectious agents whose presence would render the sample
unsuitable for use. For the diagnosis of specific infectious
agents, appropriate mixtures of peptides are required in order to
obtain accurate determinations. Antibodies to individual viral
antigens derived from one or more infectious agents may be
individually detected and identified simultaneously when use is
made of test systems in which individual peptides or mixtures of
peptides are bound to the solid phase but are physically separated
as they are, for example, in the line immunoassay, such that
individual reactions can be observed and evaluated. Such tests
require the use of an appropriate combination of peptide mixtures
to achieve the desired result.
[0360] It is frequently preferable to use mixtures of peptides
rather than a single peptide for the diagnosis of ongoing or past
infections. Since individual responses to single epitopes may be
quite variable, more reliable results are often obtained when
several immunologically important epitopes are present in the
antibody test. However, since each peptide is chemically unique, it
is frequently difficult to incorporate all of the desired peptides
into one test, particularly when the peptides are to be coated
directly onto the solid phase. Not all peptides are capable of
binding to the solid phase and the peptides in the mixture may also
exhibit very different optimal coating conditions in terms of pH,
ionic strength, and buffer composition.
[0361] To determine how well biotinylated peptides would function
in a mixture when bound to streptavidin- or avidin-coated plates,
two mixtures were made of the N-terminally biotinylated versions of
the HIV-1 peptides TM-HIV-1 (hereabove referred to as 1a.1) and
V3-mn (hereabove referred to as 1b.4), the HIV-2 peptide TM-HIV-2
(hereabove referred to as 2a), and the hepatitis C virus peptides
II, IX, and XVIII. Mixture A contained each of the six biotinylated
peptides at a concentration of 1 microgram per milliliter (6
micrograms per milliliter peptide, total) while in mixture B, each
peptide was present at a concentration of 0.1 microgram per
milliliter (0.6 microgram per milliliter peptide, total). The
individual peptides were coated at a concentration of 1 microgram
per milliliter. For purposes of comparison, mixtures A and B were
also coated directly onto the wells of a microtiter plate. Samples
from HIV-1, HIV-2, and HCV-seropositive donors were tested and
compared to sera from seronegative blood donors. A cut-off
absorbance value of 0.250 was used to determine whether a reaction
was positive or negative. Absorbance values equal or greater than
0.250 were considered positive while absorbance values below this
value were considered negative. The results of this experiment are
shown in Table 8. Based on the reactions to the individual
peptides, all of the HCV serum samples were negative for antibodies
to either HIV-1 or HIV-2. One HIV-2 sample (no. 1400) had
antibodies to HCV peptide XVIII. Of the HIV samples tested, there
was no indication of crossreactivity and the ELISA based on
individual peptides is specific. Both mixtures A and B gave good
results when bound to avidin-coated microtiter plates. As expected,
these mixtures were recognized by HIV-1, HIV-2, and HCV-positive
sera but not by sera from seronegative blood donors. In contrast,
when these mixtures were coated directly onto the microtiter
plates, the results were considerably less satisfactory, with many
samples giving a reaction which fell below the cut-off value
applied. These results serve to illustrate quite convincingly the
enhanced immunological recognition of biotinylated peptides bound
to avidin as compared to peptides coated directly onto the solid
phase as well as the advantages of using mixtures of peptides for
multiple antibody detection.
EXAMPLE 12
Use of Biotinylated Peptides for Mapping of Epitopes in
Diagnostically Useful Regions of HCV
[0362] It was demonstrated in Example 6 that several diagnostically
important regions of the HCV polyprotein, such as Core, NS4, and
NS5, can be identified using overlapping 20-mer biotinylated
peptides. Extensive serological testing identified the most useful
20-mer biotinylated peptides which permitted to develop a line
immunoassay utilizing these biotinylated peptides. However, it was
desirable to know more exactly where in these 20 amino acid-long
sequences the epitopes were located. One reason is that, if shorter
sequences could be identified, it would be possible to make
synthetic peptides containing two or three epitopes without the
peptide becoming prohibitively long.
[0363] Epitopes present in a position of the putative HCV proteins
were mapped using the method originally described by Geysen, H. M.,
Meloen, R. H., and Barteling, S. J.; Proc. Natl. Acad. Sci. USA
(1984) 81:3998-4002. Consecutive peptides nine amino acids in
length with an eight amino acid overlap were synthesized on
polyethylene pins derivatized with a non-cleavable linker. This
peptide length was chosen because it is larger than the size of
typical linear epitopes which are generally between 5 and 7 amino
acids in length. By synthesizing 9-mers, the probability that
epitopes would be missed was minimized.
[0364] The regions in the HCV polyprotein which were scanned
contain Core sequences (aa. 1 to 80), NS4 (aa. 1688 to 1755) and
NS5 (aa. 2191 to 2330). These regions correspond to the previously
determined 20-mers: Peptide I to VII (Core 1 to 13), Peptide VIII
to XIV (NS4-1 to 9), and peptide XV to XIX (NS5-13 to 33).
[0365] Following synthesis, all peptides were N-acetylated prior to
side chain deprotection in order to remove the unnatural positive
charge at the amino terminus.
[0366] The peptides were then assayed for their ability to be
recognized by antibodies present in sera from HCV seropositive
donors. The results of these experiments are shown in FIG. 6a to
6c. The optical density values shown are the average of duplicate
determinations and have been assigned to the first amino acid of
the 9-mer sequence.
[0367] The antibody binding profiles for 10 different HCV sera are
shown in FIG. 6a. It is clear that the Core protein of HCV presents
well-defined linear epitopes which are readily stimulated by
synthetic peptides. At least superficially, most sera appear to
give very similar patterns. Closer inspection, however, reveals
that there are individual differences. The various regions of the
HCV Core protein which are recognized by antibodies are perhaps
more properly termed epitopic clusters rather than epitopes as
such, since each region is undoubtedly composed of several
overlapping epitopes which are difficult, if not impossible, to
distinguish using polyclonal sera. An attempt was made to identify
core epitopes in each of the epitopic clusters. Used in this sense,
the word "core" refers to the minimal amino acid sequence
recognizable by antibodies. It should be emphasized, however, that
amino acids in addition to the core sequence may improve reactivity
particularly in the case of polyclonal sera. An analysis of the
epitopes is given in Table 9. By comparing the reactions of the
various sera, subdomains of epitopic clusters could be identified.
Some sera react predominately with one subdomain and not with
others, while other sera recognize all of the subdomains but still
allow the subdomains to be distinguished because each forms a
shoulder in the large peak which defines that particular epitopic
clusters. Table 9 and FIG. 7a shows the locations of the core
epitopes with respect to the sequences of the 20-mers.
[0368] The series of 9-mers corresponding to each of the 20-mer
Core peptides are shown in FIG. 7a together with the placement of
each of these sequences in relation to an antibody recognition
profile for one of the antisera tested.
[0369] The antigenic profiles for the NS4 protein obtained with the
10 sera are shown in FIG. 6b. In general, the reaction of these
sera with the 9-mers was less pronounced than with the peptides
from the Core protein. It was, nevertheless, still possible to
identify epitopic regions in the N-terminal sequences of the viral
NS4 protein. The core sequences of these epitopes are analyzed in
Table 10 and show their relation to the 20-mer synthetic peptides
which are diagnostically important in this region. The 9-mers
corresponding to the different 20-mers are shown in FIG. 7b
together with their placement in relation to an example of an
antigenic profile. It can be seen that the 20-mers correspond quite
well to the epitopes in this region.
[0370] The portion of the NS5 protein which was scanned corresponds
to the region covered by the 20-mer peptides 13 to 33. The
antigenic profiles obtained in this region are shown in FIG. 6c.
Again, an attempt was made to define core epitopes and these are
listed in Table 11. Little antibody binding was observed in the
amino terminal portion of this sequence. In FIG. 7c, the 9-mers
corresponding to the 20-mer peptides NS5-21 to NS5-31 are listed
and their positions are shown relative to one of the antigenic
profiles.
[0371] In particular, it is apparent that, the importance of the
sequence represented by HCV peptide XVI (NS5-27) would be severely
underestimated based on the results obtained with the overlapping
9-mers. The importance of this sequence would also be
underestimated if unbiotinylated HCV peptide XVI (NS5-27) were
evaluated in an ELISA following direct coating onto the microtiter
plate (see Table 4B). However, the biotinylated version of this
peptide when bound to streptavidin- or avidin-coated plates reveals
the presence of a very important epitope which is of diagnostic
value.
[0372] In contrast to the often weak binding observed with the
9-mers, the binding with the 20-mers was frequently quite strong
(see table 12). In several cases the differences are dramatic. For
example, serum 8241 does not recognize any of the 9-mers, whereas
the binding to the peptides HCV2 (peptide IX) and HCV5 (peptide XI)
is very strong. Moderate binding was also observed to the peptide
HCV7 (peptide XIII). This would seem to indicate that there is an
important structural component to these epitopes which is present
in the 20-mers but which is absent in the 9-mers.
EXAMPLE 13
Use of Biotinylated Peptides for Identification of Epitopes in the
N-Terminus of NS1 Region of HCV Line Immunoassay
[0373] Epitopes can also be identified using the line immunoassay
(LIA). In general, unbiotinylated peptides bind better to nylon
membranes than to polystyrene ELISA plates. Nevertheless,
biotinylated peptides complexed with streptavidin or avidin give
superior results in the line immunoassay than do their
unbiotinylated counterparts bound directly to the membrane. In
order to illustrate this, unbiotinylated and N-terminally
biotinylated versions of HCV peptides XXg-1 and XXg-2 were
synthesized. The unbiotinylated peptides were applied to the
membrane as a stock solution containing 100 micrograms per
milliliter peptide, whereas the biotinylated peptides were bound to
streptavidin and applied as a stock solution of 100 micrograms per
milliliter complex. The amount of biotinylated peptide in the stock
solution was therefore approximately 10 micrograms per milliliter.
Three human IgG control lines were also applied to the strips in
order to assist in evaluating the intensity of the reactions.
Following application of the antigen lines, excess binding sites on
the membrane were blocked with casein in phosphate-buffered saline.
The membrane was subsequently cut into strips perpendicular to the
direction in which the antigen lines were applied and the resulting
strips were incubated with a panel of sera from HCV-seropositive
donors. Bound antibody was detected visually using goat anti-human
IgG antibodies conjugated to the enzyme alkaline phosphatase after
addition of 5-bromo-4-chloro-3-indolylphosphate and Nitro Blue
tetrazolium. The results are shown in FIG. 8.
[0374] The specificity of the reactions is demonstrated by the
absence of detectable antibody binding to any of the HCV peptides
by three sera (33, 34, and 35) obtained from HCV-seronegative
donors. The reactions of sera 1 to 32 to the unbiotinylated HCV
peptides XX-1 and XX-2 are generally absent or exceedingly weak. In
contrast, many of the sera tested recognized the biotinylated
versions of these peptides when complexed to streptavidin. The
antibody reactions to the biotinylated peptides are significantly
stronger in spite of the fact that only approximately one-tenth the
amount of peptide was present in these stock solutions compared to
the amount present in the stock solutions of the unbiotinylated
peptides. The results obtained using the biotinylated peptides
demonstrate the presence of a diagnostically useful epitope in
these peptide sequences which is not evident when the
unbiotinylated versions of the peptides are used.
[0375] A total of 8 sequences spanning the hypervariable N-terminus
of the HCV E2-NS1 region (aa 383 to 416 of the HCV polyprotein) of
different HCV isolates were chosen for further evaluation. These
aligned sequences (one-letter code) are as following:
39 XXa GETYTSGGAASHTTSTLASLFSPGASQ (1) (SEQ ID NO:125) RIQLVNT XXb
GHTRVSGGAAASDTRGLVSLFSPGSAQ (2) (SEQ ID NO:126) KIQLVNT XXc
GHTRVTGGVQGHVTCTLTSLFRPGASQ (3) (SEQ ID NO:127) KIQLVNT XXd
GHTHVTGGRVASSTQSLVSWLSQGPSQ (4) (SEQ ID NO:128) KIQLVNT XXe
GDTHVTGGAQAKTTNRLVSMFASGPSQ (5) (SEQ ID NO:129) KIQLINT XXf
AETYTSGGNAGHTMTGIVRFFAPGPKQ (6) (SEQ ID NO:130) NVHLINT XXg
AETIVSGGQAARAMSGLVSLFTPGAKQ (7) (SEQ ID NO:131) NIQLINT XXh
AETYTTGGSTARTTQGLVSLFSRGAKQ (8) (SEQ ID NO:132) DIQLINT
[0376] These sequences are derived from isolates described by the
following groups:
[0377] (1) Hijikata et al., Biochem. Biophys. Res. Comm.
175:220-228, 1991.
[0378] (2) unpublished results
[0379] (3) Hijikata et al., Biochem. Biophys. Res. Comm.
175:220-228, 1991.
[0380] (4) Kato et al., Proc. Natl. Acad. sci. USA 87:9524-9528,
1990.
[0381] (5) Takamizawa et al., J. Virology 65:1105-1113, 1991.
[0382] (6) Weiner et al., Virology 180:842-848, 1991.
[0383] (7) Okamoto et al., Japan. J. Exptl. Med. 60:167-177,
1990.
[0384] (8) Kremsdorfl et al., Abstract. V64, Third International
Symposium on HCV, Strasbourg, France, September, 1991.
[0385] Since the sequences are rather long and because secondary
structure-related difficulties were predicted to occur during
synthesis, it was decided to split the sequences into two
overlapping parts (a=amino acid 383 to 404 and b=amino acid 393 to
416 of the HCV polyprotein). Subdividing the sequence also allows
the position of the epitope to be move accurately defined.
[0386] All of the peptides were N-terminally biotinylated,
complexed with streptavidin and used to prepare LIA-strips (data
not shown)
[0387] When only the LIA-positive samples are considered, the
detection rate on the E2/NS1 peptides was found to be on the order
of 90 percent. The correlations between recognition of the E2/NS1
peptides and LIA reactivity as well as the scores for the
individual peptides are shown in Table 13. It was also clear from
the observed reactions that the primary epitope in this sequence is
located towards the carboxy-terminus of the hypervariable region.
There were exceptions to this. Each serum appeared to have its own
recognition pattern which underscores the importance of using a
mixture of different sequences if this epitope is to be included as
a line in the LIA. It would also appear that either there is a
considerable degree of crossreactivity between the type 1a and type
1b sequences, or that most people are doubly infected. It is a
simple matter to distinguish between these two possibilities by
selectively removing the antibodies, which bind to one sequence,
and looking to see what the effect is on antibody recognition of
the other sequences. A number of samples gave a rather weak
reaction to one or more E2/NS1 peptides but were LIA negative.
While most probably false positive reactions, these sera may also
be from people who were previously infected but who have resolved
the infection.
EXAMPLE 14
Use of Combined HCV Peptides from the Core Region of HCV for the
Detection of Antibodies by LIA
[0388] In order to reduce the overall number of peptides in a HCV
ELISA or LIA, biotinylated peptides can be synthesize which span
other immunologically important peptides. Examples of such
"combined" HCV peptides from the core protein NS3 region of HCV are
given below:
40 Peptide Sequence core 1 M S T I P K P Q R K T K R N T N R R P Q
(SEQ ID (I) NO:133) core 2 P K P Q R K T K R N T N R R P (SEQ ID
(II) NO:134) core 3 R N T N R R P Q D V K F P G G G Q I V G (SEQ ID
(III) NO:135) core M S T I P K P Q R K T K R N T N R R P Q D V K F
P G G G Q I V G (SEQ ID 123 NO:136) core 6 V G G V Y L L P R R G P
R L G V R A T R (SEQ ID (IVa) NO:137) core 7 L P P R G P R L G V R
A T R K T S E R S (SEQ ID (IV) NO:138) core 9 T R K T S E R S Q P R
G R R Q P I P K V (SEQ ID (V) NO:139) core R S Q P P G R R Q P I P
K V R R P E G P (SEQ ID 10 NO:140) (VI) core G G V Y L L P R R G P
R L G V R A T R K T S E R S Q P R G R R Q P I P K V R R (SEQ ID
7910 NO:141)
[0389] All of these peptides have been provided with a Gly-Gly
spacers and a biotin at the amino terminus. The peptides were
evaluated in a line immunoassay experiment (LIA) and compared to
the shorter core peptides. The results are shown in FIG. 9. The
longer core peptides compare very favorably to the shorter peptides
and consistently give a more intense reaction. This is could be
explained if (i) the longer peptides incorporate two or more
epitopes which were previously spread over two separate peptides
and/or (2) there is any conformational contribution which may be
more prominent in the longer peptides.
EXAMPLE 15
Use of Combined HCV Peptides from the NS4 and NS5 Regions of HCV
for the Detection of Antibodies by LIA
[0390] Other peptides combine sequences in NS4 and NS5 which are as
following:
41 Peptide Sequence NS4-5 (XI) S Q H L P Y I E Q G M M L A E Q F K
Q K (SEQ ID NO:142) NS4-7 (XIII) L A E Q F K Q K A L G L L Q T A S
R Q A (SEQ ID NO:143) NS4-57 S Q H L P Y I E Q G M M L A E Q F K Q
K A L G L L Q T A S R Q A (SEQ ID NO:144) NS5-25 (XV) E D E R E I S
V P A E I L R K S R R F A (SEQ ID NO:145) NS5-27 (XVI) L R K S R R
F A Q A L P V W A R P D Y N (SEQ ID NO:146) NS5-2527 E D E R E I S
V P A E I L R K S R R F A Q A L P V W A R P D Y N (SEQ ID
NO:147)
[0391] The general advantage in using the longer peptides lies in
the fact that their use in an ELISA or LIA leaves more space for
the incorporation of other peptides carrying immunologically
important epitopes.
EXAMPLE 16
Use of Type-Specific HCV NS4 Peptides for the Detection of
Antibodies by LIA
[0392] Equivalent peptides containing HCV type 2 and type 3 NS4
sequences which correspond to the type 1 peptides found to contain
epitopes in NS4 were synthesized. The sequences of these peptides
are shown below for comparison:
42 Pep- tide Sequence NS4-1 L S G K P A I I P D R E V L Y R E F D E
(1) (SEQ ID NO:148) NS4-1 V N Q R A V V A P D K E V L Y E A F D E
(2) (SEQ ID NO:149) NS4-5 S Q H L P Y I E Q G M M L A E Q F K Q K
(1) (SEQ ID NO:150) NS4-5 A S R A A L I E E G Q R I A E M L K S K
(2) (SEQ ID NO:151) NS4-7 L A E Q F K Q K A L G L L Q T A S R Q A
(1) (SEQ ID NO:152) NS4-7 I A E M L K S K I Q G L L Q Q A S K Q A
(1) (SEQ ID NO:153)
[0393] LIA strips were prepared using these nine peptides which
were subsequently incubated with different sera. The results are
shown in FIG. 10. Two of the sera which were previously negative on
type 1 NS4 peptides gave a positive reaction to the type 3 and type
2 peptides. This indicates that it is possible to increase the NS4
detection rate using these peptides.
EXAMPLE 17
Use of Biotinylated Peptides from the V3 Loop Region of GP120 of
Different HIV-1 Isolates in a Line Immunoassay for the Detection of
HIV Antibodies
[0394] In order to determine the general diagnostic value of the V3
loop region of gp120, nine peptides derived from this region of
nine different HIV-1 isolates were synthesized and included in a
LIA. All nine peptides were provided with a Gly-Gly spacer and an
N-terminal biotin. The aligned peptides (one-letter amino acid
code) sequences are as following:
43 CON NNTRKSIHI-- 23 (SEQ ID NO:154) GPGRAFYTTGEIIG SF2
NNTRKSIYI-- 23 (SEQ ID NO:155) GPGRAFHTTGRIIG SC NNTTRSIHI-- 23
(SEQ ID NO:156) GPGRAFYATGDIIG MN YNKRKRIHI-- 23 (SEQ ID NO:157)
GPGRAFYTTKNIIG RF NNTRKSITK-- 23 (SEQ ID NO:158) GPGRVIYATGQIIG MAL
NNTRRGIHF--GPGQALYTTG- 22 (SEQ ID NO:159) IVG BH
NNTRKSIRIQRGPGRAFVTIGKI-G 24 (SEQ ID NO:160) ELI
QNTRQRTPI--GLGQSLYTT-RSRS 22 (SEQ ID NO:161) ANT70
QIDIQEMRI--GP-MAWYSMG-IGG 21 (SEQ ID NO:162)
[0395] The peptides were mixed with streptavidin in a slight molar
excess over biotin binding sites and the peptide: streptavidin
complexes were separated from unbound material over Sephadex G-25.
Material eluting in the void volume was used in the preparation of
the LIA.
[0396] A total of 332 sera were tested which had been obtained from
various geographical regions. Since it is known that virus strains
isolated in Europe or North America exhibit less strain-to-strain
variability than African isolates, geographical differences in the
V3-loop sequence recognition were to be expected. The reactions of
the various lines were evaluated as positive (i) or negative (o),
data not shown.
[0397] A complete evaluation of the sera is given in Table 14. In
total, 307 of the 332 sera gave a reaction to at least one peptide
on the V3-loop LIA. Those sera which failed to give a reaction to
any peptide on the V3-loop LIA were tested by Western Blot to
determine whether the sera were indeed positive for anti-HIV-I
antibodies. It was found that 6 sera were in fact negative. The
total number of positive sera tested was therefore 326. There were,
however, 19 sera which contained antibodies to gp120 which failed
to react with any of the V3-loop LIA, the percentage of sera giving
a positive reaction was, in global terms, 94%. There were, however,
significant geographical differences. These differences are shown
in Table 15.
[0398] The total percentage of sera from the different geographical
regions giving at least one positive reaction can be summarized as
follows:
44 European 100% African 94% Brazilian 92%
[0399] Additional evaluations with European samples indicate that
this percentage is, in fact, some what less than 100% (data not
shown). African samples which failed to give a reaction in the LIA
have not been tested by Western Blot to confirm the presence of
other HIV antibodies.
[0400] That the European sera would score well was expected. The
lower score obtained for the African sera was also not totally
unexpected, since it is known that there is more viral
heterogeneity in Africa. Since V3-loop sequences of African strains
of HIV have not been as extensively characterized as the European
or North American strains, it is clear that we either do not have a
representative sequence, or that attempting to characterize African
strains in terms of a consensus sequence is not possible exercise
since there is too much sequence divergence. The results obtained
with the Brazilian sera were unexpected since nothing has ever been
reported concerning HIV variability in Brazil. From these results,
it appears that the situation in Brazil more closely resembles the
situation in Africa and not the situation in North America or
Europe.
EXAMPLE 18
Improved Detection of HIV-1 Anti-V3 Domain Antibodies in Brazilian
Sera Using a V3 Sequence Derived from a Brazilian Isolate
[0401] Brazilian serum samples which failed to recognize any HIV-1
V3 loop sequences present on the previously described LIA strips
but which were positive for antibodies which recognized the HIV-1
gp120 protein on Western blots were selected for further study. In
one of these samples, V3 loop sequences of virus present in the
serum sample could be amplified using the polymerase chain reaction
using primers derived from the more constant regions flanking the
hypervariable domain. The resulting DNA fragment was subsequently
cloned and the nucleotide sequence was determined. A peptide
corresponding to the deduced amino acid sequence encoded by this
fragment was synthesized and tested for its ability to be
recognized by various HIV-1 antibody-positive sera. The sequence of
this peptide was as follows:
[0402] Peptide V3-368:
45 Asn Asn Thr Arg Arg Gly Ile His (SEQ ID NO:163) Met Gly Trp Gly
Arg Thr Phe Tyr Ala Thr Gly Glu Ile Ile Gly
[0403] A spacer consisting of two glycine residues was added to the
amino terminus. Thereafter, the resulting N-terminal glycine
[0404] residue was biotinylated. The ability of European, African,
and Brazilian HIV-1 antibody-positive sera to recognize this
peptide was investigated and compared to the ability of these same
sera to recognize the consensus sequence peptide in an ELISA. The
two peptides were also evaluated together as a mixture. These
results are summarized in table 16. These results demonstrate that
with sera of European or African origin, the V3-368 peptide does
hot result in an increased anti-V3 loop antibody detection over
that which is observed with the V3con peptide. In contrast, the use
of the V3-368 peptide results in a marked improvement in V3
antibody detection with Brazilian sera. Although this peptide is
recognized less frequently than the V3con peptide, the two peptides
complement each other to raise the detection rate from 83.3 percent
using the V3con peptide alone to 97.2 percent when the two peptides
are used together.
EXAMPLE 19
Antibody Recognition of HIV-2 V3 Loop Sequences
[0405] The outer membrane glycoprotein of HIV-2 (gp105) is similar
to that of HIV-1 with respect to its organization. Like the gp120
protein of HIV-1, the gp105 protein of HIV-2 consists of domains of
variable sequence flanked by domains of relatively conserved amino
acid sequence. In order to detect antibodies specific for the V3
domain of HIV-2 produced in response to infection by this virus,
biotinylated peptides were synthesized corresponding to the V3
sequences of the HIV-2/SIV isolates GB12 and isolate SIV mm 239
(Boeri, E., Giri, A., Lillo, F. et al.; J. Virol. (1992)
66(7):4546-4550). The sequences of the peptides synthesized are as
follows:
[0406] V3-GB12:
46 Asn Lys Thr Val Val Pro Ile (SEQ ID NO:164) Thr Leu Met Ser Gly
Leu Val Phe His Ser Gln Pro Ile Asn Lys
[0407] V3-239:
47 Asn Lys Thr Val Leu Pro Val Thr (SEQ ID NO:165) Ile Met Ser Gly
Leu Val Phe His Ser Gln Pro Ile Asn Asp
[0408] Two glycine residues were added at the N-terminus of each
peptide to serve as a spacer and a biotin was coupled to the
a-amino group of the resulting N-terminal glycine. The peptides
were bound to streptavidin and coated in the wells of microwell
plates. HIV-2 antibody-positive sera were used to evaluate these
two peptides in an ELISA. These results are summarized in Table 17.
The results clearly demonstrate the usefulness of these two peptide
sequences for the diagnosis of HIV-2 infection.
EXAMPLE 20
Localization of the Epitope at the Carboxy Terminus of C-100 with
Biotinylated Peptides
[0409] There have been various reports of an epitope located
towards the carboxy-terminal portion of the C-100 protein (EP-A-0
468 527, EP-A-0 484 787). Reactivity of certain sera toward this
epitope and not to epitopes located within the 5-1-1 fragment could
explain why these sera give a positive reaction on C-100 but not to
the above-described peptides (described in the above-mentioned
examples. The five overlapping biotinylated peptides synthesized
NS4-a, b, c, d and e are shown in FIG. 11 and cover the
carboxy-terminus of C-100 except for the last three amino acids.
LIA strips prepared with these peptides were tested using a series
of HCV Ab-positive and negative sera. The results of this
experiment (data not shown) are summarized below:
48 peptide Nr. of reactive sera Percentage NS4-a 0 0% NS4-b 2 3%
NS4-c 0 0% NS4-d 0 0% NS4-e 16 27%
EXAMPLE 21
Use of Biotinylated Hybrid Peptides Containing Epitopes from
Different HCV Proteins
[0410] A fine mapping of the epitopes in the immunologically most
important regions of the HCV polyprotein using 9-mers was performed
as illustrated in Example 12. Using this information, 3 peptide
sequences were devised which consisted of three 9-mer stretches of
HCV sequence separated by 2 amino acid-long spacers. In general,
Gly-Gly, Gly-Ser or Ser-Gly spacers were used to provide chain
flexibility. The arrangement of the epitopes in the three hybrid
peptides synthesized and their sequences are shown in FIG. 12. The
three peptides were evaluated on a LIA strip. In the first
evaluation, the sera originally used for the epitope fine mapping
experiments were used since the precise interactions of these sera
with the epitopes is known. These results are shown in FIG. 13 and
are summarized in Table 16. The order in which the epitopes were
incorporated into these three hybrid peptides was arbitrary. It is
advantageous, however, to link the epitopes together in a limited
number of peptide chains rather than attempting to develop a test
based on individual 9-mers. The use of separate 9-mers would
rapidly saturate the streptavidin binding sites on the plate (one
biotin binding site/9-mer) whereas incorporating the 9-mers into a
limited number of peptides as was done in these experiments would
enable one to bind 3 times as much (one biotin binding site/three
9-mers).
EXAMPLE 22
E2/NS1 "b" Sequence Mixotope Peptides
[0411] The results using synthetic peptides (see Examples above)
have indicated that most HCV seropositive sera contain antibodies
directed towards the hypervariable N-terminus of E2/NS1. However,
because of the hypervariable nature of this region of the protein,
it is necessary to use a rather wide spectrum of sequences in order
to detect these antibodies in an acceptably high percentage of
sera. Analysis of available sequences revealed that the observed
amino acid substitutions were not entirely random and that certain
amino acids were preferred in certain positions within the
sequence. Since the hypervariable sequence is rather long, this
sequence who divided into two overlapping portions ("a" and "b") to
improve the quality of the product and simplify the synthesis.
Subdividing this region also permitted the determination of that
the portion of this N-terminal segment of the E2/NS1 protein which
was most frequently recognized by antibodies was located in the
region encompassed by the "b" versions of these sequences. Given
the sequence information shown in FIG. 14 a "mixotope" was
synthesized which contains at each position all the amino acids
found in the naturally occurring isolates examined. The strategy
followed in the synthesis of the mixotope is depicted in FIG. 15.
The strategy for designing mixotopes is reviewed in Gras-masse et
al., Peptide Res. (1992) 5:211-216. The resin was divided into a
number of portions equal to the number of amino acids to be
coupled. The coupling reactions were carried out individually so as
to avoid problems arising due to differences in coupling kinetics
between the various amino acids. Following the coupling reactions,
the resin portions were pooled and mixed thoroughly. The total
number of variants obtained for this 23 amino acid-long sequence
was +1.147.times.1010. The increasing number of variants as a
function of chain length as measured from the carboxy-terminus or
amino-terminus is shown in FIG. 14. The rationale behind the
mixotope approach is that epitopes are composed of amino acids
whose contribution to antibody binding is not equal. Antibodies may
recognize an epitope even though there may be a relatively large
number of (generally not random) substitutions in certain
positions. In this respect, the antigenic complexity of the
mixotope should be substantially less than the number of variants
comprising the mixture. For the sake of illustration, if it is
assumed that an average epitope is 6 amino acids in length, it is
possible to calculate the number variants for each successive 6
amino acid long segment in the sequence. The number of variants as
a function of position in the sequence is shown in FIG. 14. The
actual number of functional variant sequences will be equal to the
number shown for any 6 amino acid-long sequence which happens to
correspond to an epitope, divided by a degeneracy factor equal to
the number if tolerated substitutions in each position of the
epitope but modified to reflect the degree to which the particular
substitutions are tolerated. Unfortunately, the exact position(s)
of the epitope(s) are not known. It should be stated explicitly
that this is not a random peptide library. Key positions in the
total sequence which do not tolerate substitutions, as evidenced by
the absence of amino acid variations in naturally occurring
isolates, are preserved. One disadvantage to this synthetic
approach is that rare amino acid substitutions are overrepresented
and will tend to dilute out the more commonly encountered amino
acids. On the other hand, the possibility existed that
overrepresentation of rare substitutions might allow the detection
of antibodies not detectable with epitope sequences comprised of
more frequently encountered amino acids. Following completion of
the synthesis of the mixotope, all peptide chains were provided
with a (Gly)2 spacer and a biotin to facilitate immunological
evaluation. A multiple antigen peptide (MAP) version of the
mixotope may also be synthesized in parallel. One result of
previous studies was that while approximately 90 percent of
HCV-positive sera could be shown to contain anti-E2/NS1 antibodies
directed against the N-terminal hypervariable region with the 16
"a" and "b" sequences investigated. The apparent lack of these
antibodies in the remaining 10 percent of HCV antibody-positive
sera could be due to two factors: 1) these patients fail to produce
antibodies against this portion of E2/NS1, or 2) has not yet been
identified the correct sequence with
[0412] which to detect these antibodies. Based on experiments with
the HIV-1 V3 loop, this latter possibility did not seem at all
unrealistic. LIA strips were prepared which contained the 8 "b"
sequences previously used in addition to the mixotope. Sera were
selected which previously scored positive on at least one of the
eight defined sequences as well as sera, which scored negative. In
total, 60 sera were tested, of which 56 previously gave a positive
reaction and 4 were previously found to be negative. Of the 56
sera, which had previously scored positive, 21 reacted with only
one or two of the peptides on the strip or only gave a very weak
reaction. (data not shown) The mixotope was; recognized by
approximately one-third of all the sera tested. The reaction of
some sera to the mixotope was surprisingly strong, however, it may
be possible that the collection of E2/NS1 sequences on which the
mixotope was based is not truly representative. It is expected that
the mixotope MAP will elicit the production of broad specificity
antisera directed against the amino-terminus of E2/NSI.
EXAMPLE 23
Use of Branched HCV N-Terminal E2/NS1 Region Peptides for Raising
Antibodies
[0413] Several sequences from the N-terminus of E2/NS1 were
selected for synthesis as multiple antigen peptides (MAP's) using
the technique described by Tam (Proc. Natl. Acad.
[0414] Sci. USA 85:5409-5413,1988). The strategy used to synthesize
the branched peptides is shown schematically in FIG. 16. Rabbits
(two for each MAP) were given an initial injection and were boosted
once before blood was drawn for a first evaluation of antibody
production. The antisera were tested on LIA strips containing a
total of 16 E2 peptides (sequences derived from 8 type 1 isolates,
"a" and "b" versions of each). Examination of the LIA strips
reveals that there is considerable cross-reaction between the
antibodies raised in the rabbits and the various E2 peptides on the
strips (FIG. 17). The fact that both "a" and "b" versions can be
found which are recognized by the different antisera indicates that
there is at least one epitope located in the region where these two
versions overlap.
EXAMPLE 24
Diagnosis of HTLV Infection Using Biotinylated Synthetic
Peptides
[0415] HTLV-I and II are antigenically related members of a family
of oncogenic retroviruses. HTLV-I infection has been shown to be
associated with two disease syndromes:
[0416] HTLV-1-associated myelopathy/tropical spastic paraparesis
(neurological disorders) and adult T-cell leukemia (ATL). In
contrast, HTLV-II has not been conclusively linked to any known
disease syndrome. This virus was originally isolated from a patient
with hairy cell leukemia, however, no causal relationship between
HTLV-II infection and the disease state could be established. Since
HTLV-I infection has definitely been demonstrated to have the
potential to result in human disease while HTLV-II infection has
not, it is of clinical interest to be able to differentiate between
these two infectious agents. Since these two viruses are
antigenically highly related, it is difficult to discriminate
between HTLV-I and HTLV-II infections when viral or recombinant
antigens are used for antibody detection. A number of biotinylated
peptides were synthesized and evaluated for their ability to detect
antibodies raised in response to infection by either HTLV-I or
HTLV-II. Some of the peptides were chosen because they contain
epitopes, which are highly conserved between HTLV-I and HTLV-II and
should therefore be useful reagents for detecting HTLV infection
without regard to virus type. Still other peptides were chosen
because they contain epitopes, which should allow HTLV-I and
HTLV-II infections to be discriminated. The peptides synthesized
are as follows:
[0417] I-gp46-3:
49 Bio Gly Gly Val Leu Tyr Ser Pro Asn Val Ser Val Pro Ser Ser (SEQ
ID NO:166) Ser Ser Thr Leu Leu Tyr Pro Ser Leu Ala
[0418] I-gp46-5:
50 Bio Gly Gly Tyr Thr Cys Ile Val Cys Ile Asp Arg Ala Ser Leu (SEQ
ID NO:167) Ser Thr Trp His Val Leu Tyr Ser Pro
[0419] I-gp46-4:
51 Bio Gly Gly Asn Ser Leu Ile Leu Pro Pro Phe Ser Leu Ser Pro (SEQ
ID NO:168) Val Pro Thr Leu Gly Ser Arg Ser Arg Arg
[0420] I-gp46-6:
52 Bio Gly Gly Asp Ala Pro Gly Tyr Asp Pro Ile Trp Phe Leu Asn (SEQ
ID NO:169) Thr Glu Pro Ser Gln Leu Pro Pro Thr Ala Pro Pro Leu Leu
Pro His Ser Asn Leu Asp His Ile Leu Glu
[0421] I-p21-2:
53 Bio Gly Gly Gln Tyr Ala Ala Gln Asn Arg Arg Gly Leu Asp Leu (SEQ
ID NO:170) Leu Phe Trp Glu Gln Gly Gly Leu Cys Lys Ala Leu Gln Glu
Gln Cys Arg Phe Pro
[0422] I-p19:
54 Bio Gly Gly Pro Pro Pro Pro Ser (SEQ ID NO:171) Ser Pro Thr His
Asp Pro Pro Asp Ser Asp Pro Gln Ile Pro Pro Pro Tyr Val Glu Pro Thr
Ala Pro Gln Val Leu
[0423] II-gp52-1:
55 Bio Gly Gly Lys Lys Pro Asn Arg (SEQ ID NO:172) Gln Gly Leu Gly
Tyr Tyr Ser Pro Ser Tyr Asn Asp Pro
[0424] II-gp52-2:
56 Bio Gly Gly Asp Ala Pro Gly Tyr (SEQ ID NO:173) Asp Pro Leu Trp
Phe Ile Thr Ser Glu Pro Thr Gln Pro Pro Pro Thr Ser Pro Pro Leu Val
His Asp Ser Asp Leu Glu His Val Leu Thr
[0425] II-gp52-3:
57 Bio Gly Gly Tyr Ser Cys Met Val (SEQ ID NO:174) Cys Val Asp Arg
Ser Ser Leu Ser Ser Trp His Val Leu Tyr Thr Pro Asn Ile Ser Ile Pro
Gln Gln Thr Ser Ser Arg Thr Ile Leu Phe Pro Ser
[0426] II-p19:
58 Bio Gly Gly Pro Thr Thr Thr Pro (SEQ ID NO:175) Pro Pro Pro Pro
Pro Pro Ser Pro Glu Ala His Val Pro Pro Pro Tyr Val Glu Pro Thr Thr
Thr Gln Cys Phe
[0427] A number of these peptides were used to prepare LIA strips
for the detection of antibodies to HTLV. Several of the peptides,
such as I-p19 and I-gp46-4, which are derived from regions of the
HTLV-1 p19 gag protein and envelope glycoprotein, respectively, are
expected to be recognized by antibodies produced as a result of
both HTLV-I and HTLV-II infection since these sequences are highly
homologous in the two viruses. Others, such as I-gp46-3, I-gp46-6
for HTLV"I, and II-gp52-1, II-gp52-2 and II-gp52-3 for HTLV-II may
be useful for detection of antibodies as well as discrimination.
Since there is some homology between the HTLV-I and HTLV-II
sequences, cross-reactions are to be expected. Nevertheless, the
intensities of the reactions to the various peptides should reveal
the identity of the virus to which the antibodies were
produced.
[0428] An example of LIA strips prepared with a number of the
biotinylated HTLV-I and HTLV-II peptides is shown in figure XXX.
The LIA strips were evaluated using a commercially available serum
panel (Boston Biomedica Inc., mixed titer panel, PRP203). The test
results are in complete agreement with the analysis provided by
distributor. Only one sample (nr. 9) is positive for HTLV-I.
[0429] Sample nr.12 is detected as positive because of the positive
reaction to the peptide I-p19. This sample could not be
differentiated using these peptides, nor could this sample be
differentiated by any other test used by the distributor of the
serum panel. Sample nr. 11 was found to be negative and all other
samples were found to be positive for HTLV-II. In an additional
experiment, an ELISA was performed using all 10 of the biotinylated
HTLV-I and HTLV-II peptides. The peptides were complexed with
streptavidin individually and then mixed prior to coating. Some of
the samples from the panel used to evaluate the LIA strips were
used to evaluate the peptides in the ELISA. These results are shown
in table. The ELISA in this configuration cannot be used to
differentiate HTLV-I and -II infections but should identify
HTLV-positive samples in general regardless of virus type. The
results further demonstrate the utility of these peptides for the
diagnosis of HTLV-infection.
59TABLE 1 Antibody recognition of biotinylated and unbiotinylated
HIV-1 and HIV-2 peptides Serum TM-HIV-1 TM-HIV-1 Bio TM-HIV-2
TM-HIV-2 Bio HIV-1 positive 0724 0.174 2.570 0.000 0.000 mm 0.051
2.579 0.000 0.000 YEMO 0.162 2.357 0.000 0.000 PL 0.000 1.559 0.000
0.000 VE 0.052 2.551 0.000 0.000 HIV-2 positive 1400 0.000 0.000
0.000 1.982 AG 0.000 0.000 0.000 2.323 53-3 0.000 0.000 0.000 2.365
Seronegative donors 194 0.000 0.000 0.000 0.000 195 0.000 0.000
0.000 0.000 180 0.000 0.005 0.000 0.000 204 0.000 0.001 0.000
0.000
[0430]
60TABLE 2 Comparison of antibody recognition of biotinylated and
unbiotinylated peptides from the V3 sequence of isolate HIV-1 mn.
Sample identity V3-mn V3-mn Bio Negative control 0.063 0.069 Blank
0.053 0.051 YS 1.442 2.784 DV 1.314 2.881 VE 1.717 overflow* OOST 6
1.025 2.855 OOST 8 1.389 overflow* 3990 1.442 overflow* PL 0.531
2.351 MM 0.791 2.542 4436 0.388 2.268 4438 0.736 2.554 266 0.951
2.591 OOST 4 1.106 overflow* *Absorbance value greater than
3.000
[0431]
61TABLE 3 Comparison of antibody recognition of the biotinylated
V3-mn peptide bound to streptavidin and avidin Serum Streptavidin
Avidin YS 1.236 1.721 DV 1.041 1.748 PL 0.222 0.983 3990 1.391
1.854 VE 1.526 1.908 4436 0.596 1.519 Control 0.050 0.063
[0432]
62TABLE 4 Comparison of antibody recognition of biotinylated und
unbiotinylated HCV peptides. Table 4A Antibody binding to HCV
peptide XI Serum Unbiotinylated peptide XI Peptide XI 2 0.090 1.971
3 0.443 2.086 4 0.473 1.976 6 0.053 0.518 8 1.275 2.624 10 0.764
2.321 11 0.569 2.378 23 0.775 2.503 31 0.497 2.104 77 0.093 0.159
33 0.832 1.857 49 0.515 2.180 negative serum 0.053 0.095 Table 4B:
Antibody binding to HCV peptide XVI Serum Unbiotinylated peptide
XVI Peptide XVI 1 1.038 2.435 2 0.616 1.239 6 0.100 1.595 8 0.29
1.599 10 1.033 2.847 26 0.053 1.522 83 0.912 2.221 88 1.187 2.519
89 0.495 1.530 91 0.197 2.169 95 0.109 1.484 99 0.814 2.045 100
0.474 1.637 104 0.205 0.942 105 0.313 2.186 110 0.762 1.484 111
0.193 1.465 112 0.253 1.084 113 0.833 2.535 116 0.058 1.918 120
0.964 2.332 11476 0.068 2.197 24758 0.071 0.062 266 0.712 2.262
8247 0.059 0.618 negative serum 0.063 0.067 Table 4C: Antibody
binding to HCV peptide II Serum Unbiotinylated peptide II Peptide
II 8241 0.444 0.545 8242 1.682 2.415 8243 2.181 2.306 8247 1.518
1.975 8250 0.110 0.357 8271 0.912 1.284 8273 2.468 2.769 8274 2.700
2.943 8275 1.489 2.030 8276 2.133 2.348 8277 1.771 2.572 8278 1.907
2.022 negative serum 0.047 0.070 Table 4D Antibody binding to HCV
peptide III Serum Unbiotinylated peptide III Peptide III 8241 1.219
2.066 8242 1.976 2.197 8243 1.859 2.368 8247 1.072 2.398 8248 2.742
2.918 8250 2.471 2.626 8271 1.471 2.066 8272 2.471 2.638 8273 1.543
2.697 8274 2.503 2.905 8275 1.595 2.640 8276 1.976 2.674 8277 0.735
2.327 negative serum 0.050 0.06 Table 4E Antibody binding to HCV
peptide V Serum Unbiotinylated peptide V Peptide V 8272 0.589 1.220
8273 0.294 1.026 8274 1.820 2.662 8275 1.728 1.724 8276 2.194 2.616
8277 0.770 1.796 8278 1.391 1.746 8284 0.040 0.757 negative serum
0.047 0.070 Table 4F Antibody binding to HCV peptide IX Serum
Unbiotinylated peptide IX Peptide IX 8315 2.614 2.672 8316 0.133
0.367 8317 0.855 1.634 8318 1.965 2.431 8320 0.721 0.896 8321 0.283
0.457 8326 2.219 2.540 negative serum 0.052 0.005 Table 4G Antibody
binding to HCV peptide XVIII Serum Unbiotinylated peptide XVIII
Peptide XVIII 79 1.739 2.105 83 1.121 1.232 88 0.972 1.858 89 2.079
2.309 91 2.202 2.132 99 1.253 1.526 104 1.864 1.998 105 1.522 2.053
110 1.981 2.065 111 1.363 1.542 112 1.172 1.408 116 1.534 1.978 120
1.599 2.031 1 2.523 2.691 33 1.463 1.813 39 0.068 0.213 47 2.117
2.611 negative serum 0.001 0.001
[0433]
63TABLE 4F Antibody binding to HCV peptide IX Serum Unbiotinylated
peptide IX Peptide IX 8315 2.614 2.672 8316 0.133 0.367 8317 0.855
1.634 8318 1.965 2.431 8320 0.721 0.896 8321 0.283 0.457 8326 2.219
2.540 negative serum 0.052 0.005
[0434]
64TABLE 4G Antibody binding to HCV peptide XVIII Serum
Unbiotinylated peptide XVIII Peptide XVIII 79 1.739 2.105 83 1.121
1.232 88 0.972 1.858 89 2.079 2.309 91 2.202 2.132 99 1.253 1.526
104 1.864 1.998 105 1.522 2.053 110 1.981 2.065 111 1.363 1.542 112
1.172 1.408 116 1.534 1.978 120 1.599 2.031 1 2.523 2.691 33 1.463
1.813 39 0.068 0.213 47 2.117 2.611 negative serum 0.001 0.001
[0435]
65TABLE 5 Peptideconcen- tration* coatingmeth- 3.0 1.0 0.3 0.1 0.03
0.01 od** 1 2 1 2 1 2 1 2 1 2 1 2 Unbiotinylated HCV peptide II and
HCV peptide II sample positive 8320 2.718 2.278 2.684 2.163 2.684
2.004 2.718 1.828 2.757 1.272 2.519 0.479 8242 1.427 0.539 1.368
0.408 1.365 0.234 1.399 0.058 1.481 0.048 1.196 0.051 8243 1.668
1.341 1.652 1.221 1.608 0.831 1.639 0.181 1.597 0.057 1.088 0.056
8318 2.016 0.791 1.993 0.626 1.958 0.347 2.001 0.181 2.181 0.095
2.002 0.048 sample negative 1747 0.064 0.049 0.071 0.046 0.046
0.041 0.045 0.044 0.045 0.043 0.045 0.041 1781 0.057 0.053 0.055
0.053 0.051 0.045 0.047 0.046 0.049 0.053 0.053 0.046
Unbiotinylated HCV peptide IX and HCV peptide IX sample positive
8320 1.779 0.129 0.802 0.093 1.798 0.122 1.244 0.063 1.007 0.057
0.461 0.059 8326 2.284 0.084 2.271 0.068 2.271 0.078 2.284 0.068
2.193 0.051 1.812 0.049 8242 0.791 0.059 0.777 0.052 0.795 0.048
0.911 0.046 0.496 0.047 0.215 0.049 8243 1.959 0.063 1.953 0.053
1.892 0.051 1.834 0.051 1.421 0.051 0.639 0.054 sample negative
1747 0.051 0.046 0.049 0.046 0.046 0.044 0.042 0.045 0.044 0.045
0.043 0.045 1781 0.053 0.053 0.051 0.052 0.051 0.051 0.047 0.052
0.048 0.049 0.049 0.051 Unbiotinylated HCV peptide XVIII and HCV
peptide XVIII sample positive 8326 2.315 0.052 2.331 0.053 2.331
0.053 2.331 0.049 2.219 0.051 1.848 0.051 8242 0.749 0.053 0.839
0.049 0.873 0.048 0.946 0.047 1.188 0.049 1.185 0.048 8243 0.671
0.057 0.627 0.053 0.629 0.054 0.661 0.051 0.611 0.053 0.462 0.053
8318 2.391 0.051 2.396 0.045 2.392 0.047 2.409 0.047 2.308 0.047
1.711 0.048 sample negative 1747 0.047 0.048 0.042 0.045 0.061
0.046 0.044 0.045 0.058 0.044 0.042 0.047 1781 0.053 0.055 0.048
0.054 0.048 0.051 0.048 0.051 0.051 0.051 0.045 0.053 *in
microgramsper milliliter **1. biotinylatedpeptide on
streplavidincoated plate 2. unbiotinylatedpeptidecoated
directly
[0436]
66TABLE 6 Comparison of N- and C-terminally biotinylated TM-HIV-1
peptide. TM-HIV-1 TM-HIV-1 Serum C-terminal biotin N-terminal
biotin HIV positive VE 2.079 2.240 OOST 6 1.992 2.003 MM 2.097
2.308 0724 2.322 2.291 DV 0.903 1.579 PL 1.893 1.849 2049 1.780
2.058 3990 1.959 1.870 4438 1.622 1.697 4436 2.190 2.110 OOST 7
1.728 2.027 OOST 8 2.117 2.237 OOST 9 2.119 2.222 VCM 2.131 2.263
1164 1.865 1.919 1252 2.244 2.356 0369/87 2.059 2.042 Seronegative
1784 0.000 0.000 blood donors 1747 0.000 0.000 1733 0.014 0.000
[0437]
67 TABLE 7 HCV peptide I carboxy- biotinyl- ated (bound to HCV
peptide I streptavidin- (coated directly) coated wells) HCV
antibody- positive sera 8316 2.394 2.541 8318 2.385 2.404 8320
2.760 2.762 8326 0.525 1.775 8329 2.633 2.672 8333 2.143 2.545 8334
2.271 2.549 8336 1.558 2.016 8344 1.878 2.010 8248 2.042 2.493 8244
0.077 1.399 8243 2.211 2.541 8242 1.367 2.389 8364 2.705 2.705 8374
1.070 2.151 8378 2.161 2.531 8330 1.985 2.651 8387 1.427 2.628 HCV
antibody- negative sera F88 0.000 0.026 F89 0.017 0.001 F76 0.000
0.022 F136 0.006 0.002 F8 0.000 0.000
[0438]
68TABLE 8 Use of mixtures of biotinylated peptides for antibody
detection. TM- HCV HCV HCV Mixture Mixture TM-HIV- HIV-2- peptide
peptide peptide Mixture Mixture A B 1-BIO BIO V3-mn-Bio II-BIO
IX-BIO XVIII-BIO A B Direct Direct Serum Avidin Avidin Avidin
Avidin Avidin Avidin Avidin Avidin coating coating HCV 8243 0.108
0.109 0.114 1.430 1.213 0.118 1.590 1.638 0.542 0.184 8247 0.042
0.048 0.052 1.356 0.756 0.046 0.840 1.149 0.049 0.049 8248 0.043
0.046 0.048 2.287 0.047 0.905 1.859 2.154 0.407 0.064 8269 0.053
0.049 0.056 1.213 0.051 1.513 0.923 1.268 0.078 0.067 8290 0.045
0.047 0.050 0.060 0.048 2.323 1.210 1.761 0.559 0.717 8278 0.046
0.045 0.053 1.878 0.074 0.052 1.806 1.944 0.540 0.152 8273 0.053
0.050 0.056 2.017 0.053 0.052 2.037 2.113 0.773 0.185 8285 0.134
0.163 0.143 1.592 0.270 0.146 1.746 1.822 0.908 0.401 8291 0.048
0.050 0.053 1.539 0.052 0.049 1.591 1.809 0.335 0.098 HIV-2 AG
0.054 2.065 0.068 0.081 0.064 0.058 1.833 1.880 0.054 0.056 1400
0.051 1.781 0.055 0.121 0.052 1.362 1.692 2.031 0.214 0.326 HIV-1
YS 0.046 0.046 2.201 0.048 0.049 0.049 2.045 1.845 0.200 0.052 PL
1.974 0.051 1.321 0.052 0.056 0.052 1.587 1.776 0.052 0.055 DV
1.329 0.048 2.340 0.047 0.049 0.047 1.969 1.742 0.100 0.049 3990
1.602 0.054 2.319 0.054 0.066 0.056 2.217 1.926 0.390 0.081 Blood
1785 0.046 0.047 0.048 0.045 0.050 0.047 0.047 0.049 0.045 0.049
donor 1794 0.124 0.090 0.091 0.153 0.098 0.104 0.152 0.161 0.050
0.058 1784 0.044 0.046 0.046 0.045 0.050 0.047 0.047 0.047 0.045
0.048 1782 0.052 0.057 0.059 0.057 0.062 0.053 0.057 0.059 0.049
0.056
[0439]
69TABLE 9 SEQUENCES OF THE CORE EPITOPES OF THE HCV CORE PROTEIN
HCV CORE PROTEIN AMINO ACIDS 1-90 POSITIONS OF CORE EPITOPES (SEQ
ID NO:) Epitope 1A: (SEQ ID NO:422) SEQ IDS (181-184) 15 16 (453)
(454) CORE 1 CORE 2 Epitope 1B: (SEQ ID NO:423) SEQ IDS (185-188)
17 18 (453) (454) CORE 1 CORE 2 Epitope 2: (SEQ ID NO:424) SEQ IDS
(194-199) 19 20 (454) (455) CORE 2 CORE 3 Epitope 3A: (SEQ ID
NO:425) SEQ IDS (209-212) 21 22 (456) CORE 5 Epitope 3B: (SEQ ID
NO:426) SEQ IDS (213-215) 23 24 (456) (457) CORE 5 CORE 7 Epitope
3C: (SEQ ID NO:427) SEQ IDS (218-220) 25 26 (457) CORE 7 Epitope
4A: (SEQ ID NO:428) SEQ IDS (231-233) 27 28 (458) CORE 9 Epitope
4B: (SEQ ID NO:429) SEQ IDS (236-241) 29 30 (458) (459) CORE 9 CORE
11 Epitope 5A: (SEQ ID NO:.430) SEQ IDS (248-253) 31 32 (459) (600)
CORE 11 CORE 13 Epitope 5B: (SEQ ID NO:431) (minor) SEQ IDS
(254-255) 33 34 (600) CORE 13
[0440]
70TABLE 10 SEQUENCES OF THE CORE EPITOPES OF THE HCV NS4 PROTEIN
HCV NS4 PROTEIN Positions of core epitopes (SEQ ID NO:) Epitope 1:
(SEQ ID NO:432) SEQ IDS (258-268) 35 36 (460) (461) HCV1 HCV2
Epitope 2A: (SEQ ID NO:433) SEQ IDS (281-285) 37 38 (462) (463)
(464) HCV3 HCV4 HCV5 Epitope 2B: (SEQ ID NO:434) (minor) SEQ IDS
(286-292) 39 40 (463) (464) HCV4 HCV5 Epitope 3A: (SEQ ID NO:435)
SEQ IDS (293-295) 41 42 (464) (465) (466) HCV5 HCV6 HCV7 Epitope
3B: (SEQ ID NO:436) SEQ IDS (296-300) 43 44 (465) (466) HCV6 HCV7
Epitope 4: (SEQ ID NO:437) SEQ IDS (301-317) 45 46 (466) (467) HCV7
HCV8
[0441]
71TABLE 11 SEQUENCES OF THE CORE EPITOPES OF THE HCV NS5 PROTEIN
HCV NS5 PROTEIN Positions of the core epitopes Epitope 1A: (SEQ ID
NO:438) SEQ IDS (348-349) 47 48 NS5-25 Epitope 1B: (SEQ ID NO:439)
SEQ IDS (350-352) 49 50 NS5-25 Epitope 2: (SEQ ID NO:440) SEQ IDS
(360-363) 51 52 NS5-27 Epitope 3: (SEQ ID NO:441) SEQ IDS (366-370)
53 54 NS5-29 Epitope 4: (SEQ ID NO:442) (minor) SEQ IDS (374-377)
55 56 NS5-29 Epitope 5: (SEQ ID NO:443) SEQ IDS (378-385) 57 58
NS5-31 Epitope 6: (SEQ ID NO:444) SEQ IDS (387-400) 59 60 NS5-31
NS5-33
[0442]
72TABLE 12 Antibody binding of various Core, NS4, and NS5
biotinylated 20-mers by the 10 test sera PEPTIDE ELISA (O.D.) SERUM
Core-2 Core-3 Core-7 Core-9 HCV-2 HCV-5 HCV-7 NS5-25 NS5-27 NS5-31
8242 2.415 2.197 0.632 2.315 2.114 1.625 1.252 0.268 2.318 2.453
8248 2.441 2.918 1.529 2.821 0.142 0.182 1.963 0.054 0.388 1.511
8332 1.977 2.054 1.387 1.455 0.392 0.575 0.945 0.047 2.130 2.290
8339 2.030 2.765 0.166 2.698 2.497 0.043 0.041 1.495 2.359 2.757
8358 1.982 2.135 0.357 0.685 1.779 0.623 0.598 0.069 2.249 0.182
8377 2.181 2.368 0.221 0.076 2.368 2.227 1.829 1.092 2.336 1.378
8378 2.140 2.369 1.089 1.228 1.859 1.449 2.006 0.279 1.602 2.337
8383 2.463 2.463 0.970 2.162 2.300 1.018 2.504 0.055 2.390 2.358
8241 0.545 2.066 0.448 0.274 2.421 2.280 0.968 0.050 2.456 0.273
8243 2.306 2.368 1.251 1.378 2.203 2.268 2.251 0.062 1.444
0.127
[0443]
73TABLE 13 Antibody recognition of individual E2/NS1 peptides
(percent of all sera giving positive reaction) CL14-A 7 (51) 13.7%
B 36 (51) 70.6% KATO-A 2 (51) 3.92% B 26 (51) 50.98% HCJ4-A 32 (51)
62.74% B 41 (51) 80.39% FRENCH-A 30 (51) 58.8% B 42 (51) 82.35%
YEK-A 7 (51) 13.72% B 49 (51) 96.07% TAMI-A 12 (51) 23.52% B 36
(51) 70.58% 18CH1-A 5 (51) 9.8% B 30 (51) 58.82% CHIR-A 32 (51)
62.7% B 40 (51) 78.42%
[0444]
74TABLE 14 Overall Recognition of V3-Loop Peptides CON SC MN SF2 BH
RF MAL ELI 70 Total SUM 287 261 275 258 108 146 140 24 6 COUNT 326
326 326 326 326 326 326 326 326 Gp120 positive % Reactive 88 80 84
79 33 45 43 7 2 Total SUM 287 261 275 258 108 146 140 24 6 COUNT
307 307 307 307 307 307 307 307 307 HIV-V3 positive % Reactive 93
85 90 84 35 48 46 8 2
[0445]
75TABLE 15 Recognition of Peptides According to Giographical Region
EUROPEAN % AFRICAN % BRAZILIAN % Consensus 98 Consensus 89
Consensus 82 HIV-1 (SC) 98 HIV-1 (MN) 85 HIV-1 (MN) 78 HIV-1 (SF2)
98 HIV-1 (SF2) 79 HIV-1 (SC) 75 HIV-1 (MN) 97 HIV-1 (SC) 73 HIV-1
(SF2) 72 HIV-1 (RF) 75 HIV-1 (MAL) 60 HIV-1 (RF) 38 HIV-1 68 HIV-1
(RF) 34 HIV-1 (MAL) 30 (MAL) HIV-1 (IIIB) 61 HIV-1 (IIIB) 27 HIV-1
(IIIB) 26 HIV-1 (ELI) 8 HIV-1 (ELI) 13 HIV-1 (ELI) 5 ANT 70 2 ANT
70 2 ANT 70 2
[0446]
76TABLE 16 Recognition of European, African and Brazilian HIV-1
antibody-positive sera to HIV-1 V3 loop peptides V3-con and V3-368
V3-con V3-368 V3con + V3-368 European sera number tested 36 36 36
number 33 4 33 positive number 0 12 0 negative number 3 20 3
borderline percent 92 11 92 positive percent 0 33 0 negative
percent 8 56 8 borderline African sera number tested 45 45 45
number 40 5 40 positive number 5 31 2 negative number 1 9 3
borderline percent 89 11 89 positive percent 9 69 4 negative
percent 2 20 7 borderline Brazilian sera number tested 36 36 36
number 30 16 35 positive number 1 5 1 negative number 5 15 0
borderline percent 83.3 44.4 97.2 positive percent 2.8 13.9 2.8
negative percent 13.9 41.7 0 borderline
[0447]
77TABLE 17 Recognition of HIV-2 positive sera to peptides from the
V3 loop region of HIV-2 V3-GB12 V3-239 number tested 21 21 number
positive 21 19 number negative 0 0 number borderline 0 2 percent
positive 100 90.50 percent negative 0 0 percent borderline 0
9.5
[0448]
78TABLE 18 Antibody recognition of hybrid peptides A. Discrepancies
NS4 NS5 Core LIA Serum Epitope 1 Epitope 5 Epitope 2 Epi-152 8241 +
- - + (weak) 8242 + - + + 8243 + - + + 8248 + - + + 8332 + + + +
8339 + + + + 8358 + - + + 8377 + - + + 8378 + + + + 8383 + + + + B.
NS5 NS4 Core LIA Serum Epitope 3 Epitope 3B Epitope 3A Epi-33B3A
8241 - - + - 8242 - + + + 8243 - + + + 8248 - - + + 8332 + +/- + +
8339 - - + + 8358 + - + - 8377 - + + - 8378 + + - + 8383 + + + + C.
Core Epitope NS4 NS5 LIA Serum 4B Epitope 2A Epitope 6 Epi-4B2A6
8241 - - - - 8242 + - - + 8243 - +/- - - 8248 + - - + 8332 + +/- -
+ (weak) 8339 + - + + 8358 - - - +/- 8377 - + - - 8378 + + - + 8383
+ + - +
[0449]
79TABLE 19 ANTIBODY RECOGNITION OF HTLV PEPTIDES Serum number
Optical density 1 0.303 2 3.001 3 0.644 4 1.262 6 3.001 7 2.623 9
2.607 (HTLV-I) 10 3.001 11 0.058 (negative) 13 3.001 14 3.001 15
0.850 16 0.278 19 1.048 20 3.001 21 0.805 22 0.812 23 3.001 24
0.405 25 1.521
[0450]
Sequence CWU 1
1
600 1 11 PRT Human immunodeficiency virus VARIANT (1) modified site
1 Xaa Ile Trp Gly Cys Ser Gly Lys Ile Cys Xaa 1 5 10 2 20 PRT Human
immunodeficiency virus VARIANT (1) modified site 2 Xaa Ile Trp Gly
Cys Ser Gly Lys Leu Ile Cys Thr Thr Ala Val Pro 1 5 10 15 Asn Ala
Ser Xaa 20 3 21 PRT Human immunodeficiency virus VARIANT (1)
modified site 3 Xaa Glu Arg Tyr Leu Lys Asp Gln Gln Leu Leu Gly Ile
Trp Gly Cys 1 5 10 15 Gly Lys Leu Ile Xaa 20 4 18 PRT Human
immunodeficiency virus VARIANT (1) modified site 4 Xaa Leu Gln Ala
Arg Ile Leu Ala Val Glu Arg Tyr Leu Lys Asp Gln 1 5 10 15 Leu Xaa 5
12 PRT Human immunodeficiency virus VARIANT (1) modified site 5 Xaa
Leu Trp Gly Cys Lys Gly Lys Leu Val Cys Xaa 1 5 10 6 24 PRT Human
immunodeficiency virus VARIANT (1) modified site 6 Xaa Asp Gln Gln
Leu Leu Gly Ile Trp Gly Cys Ser Gly Lys His Ile 1 5 10 15 Thr Thr
Asn Val Pro Trp Asn Xaa 20 7 24 PRT Human immunodeficiency virus
VARIANT (1) modified site 7 Xaa Asn Asn Thr Arg Lys Ser Ile His Ile
Gly Pro Gly Arg Ala Phe 1 5 10 15 Thr Thr Gly Glu Ile Ile Gly Xaa
20 8 36 PRT Human immunodeficiency virus VARIANT (1) modified site
8 Xaa Cys Thr Arg Pro Asn Asn Asn Thr Arg Lys Ser Ile His Ile Gly 1
5 10 15 Gly Arg Ala Phe Tyr Thr Thr Gly Glu Ile Ile Gly Asp Ile Arg
Gln 20 25 30 Ala His Cys Xaa 35 9 24 PRT Human immunodeficiency
virus VARIANT (1) modified site 9 Xaa Asn Asn Thr Arg Lys Ser Ile
Tyr Ile Gly Pro Gly Arg Ala Phe 1 5 10 15 Thr Thr Gly Arg Ile Ile
Gly Xaa 20 10 24 PRT Human immunodeficiency virus VARIANT (1)
modified site 10 Xaa Asn Asn Thr Thr Arg Ser Ile His Ile Gly Pro
Gly Arg Ala Phe 1 5 10 15 Ala Thr Gly Asp Ile Ile Gly Xaa 20 11 24
PRT Human immunodeficiency virus VARIANT (1) modified site 11 Xaa
Tyr Asn Lys Arg Lys Arg Ile His Ile Gly Pro Gly Arg Ala Phe 1 5 10
15 Thr Thr Lys Asn Ile Ile Gly Xaa 20 12 25 PRT Human
immunodeficiency virus VARIANT (1) modified site 12 Xaa Asn Asn Thr
Arg Lys Ser Ile Thr Lys Gly Pro Gly Arg Val Ile 1 5 10 15 Tyr Ala
Thr Gly Gln Ile Ile Gly Xaa 20 25 13 24 PRT Human immunodeficiency
virus VARIANT (1) modified site 13 Xaa Asn Asn Thr Arg Arg Gly Ile
His Phe Gly Pro Gly Gln Ala Leu 1 5 10 15 Tyr Thr Thr Gly Ile Val
Gly Xaa 20 14 26 PRT Human immunodeficiency virus VARIANT (1)
modified site 14 Xaa Asn Asn Thr Arg Lys Ser Ile Arg Ile Gln Arg
Gly Pro Gly Arg 1 5 10 15 Ala Phe Val Thr Ile Gly Lys Ile Gly Xaa
20 25 15 24 PRT Human immunodeficiency virus VARIANT (1) modified
site 15 Xaa Gln Asn Thr Arg Gln Arg Thr Pro Ile Gly Leu Gly Gln Ser
Leu 1 5 10 15 Tyr Thr Thr Arg Ser Arg Ser Xaa 20 16 23 PRT Human
immunodeficiency virus VARIANT (1) modified site 16 Xaa Gln Ile Asp
Ile Gln Glu Met Arg Ile Gly Pro Met Ala Trp Tyr 1 5 10 15 Ser Met
Gly Ile Gly Gly Xaa 20 17 25 PRT Human immunodeficiency virus
VARIANT (1) modified site 17 Xaa Asn Asn Thr Arg Arg Gly Ile His
Met Gly Trp Gly Arg Thr Phe 1 5 10 15 Tyr Ala Thr Gly Glu Ile Ile
Gly Xaa 20 25 18 38 PRT Human immunodeficiency virus VARIANT (1)
modified site 18 Xaa Arg Asp Asn Trp Arg Ser Glu Leu Tyr Lys Tyr
Lys Val Val Lys 1 5 10 15 Ile Glu Pro Leu Gly Val Ala Pro Thr Lys
Ala Lys Arg Arg Val Val 20 25 30 Gln Arg Glu Lys Arg Xaa 35 19 12
PRT Human immunodeficiency virus VARIANT (1) modified site 19 Xaa
Ser Trp Gly Cys Ala Phe Arg Gln Val Cys Xaa 1 5 10 20 22 PRT Human
immunodeficiency virus VARIANT (1) modified site 20 Xaa Lys Tyr Leu
Gln Asp Gln Ala Arg Leu Asn Ser Trp Gly Cys Ala 1 5 10 15 Phe Arg
Gln Val Cys Xaa 20 21 25 PRT Human immunodeficiency virus VARIANT
(1) modified site 21 Xaa Asn Lys Thr Val Leu Pro Ile Thr Phe Met
Ser Gly Phe Lys Phe 1 5 10 15 His Ser Gln Pro Val Ile Asn Lys Xaa
20 25 22 24 PRT Human immunodeficiency virus VARIANT (1) modified
site 22 Xaa Asn Lys Thr Val Val Pro Ile Thr Leu Met Ser Gly Leu Val
Phe 1 5 10 15 His Ser Gln Pro Ile Asn Lys Xaa 20 23 24 PRT Human
immunodeficiency virus VARIANT (1) modified site 23 Xaa Asn Lys Thr
Val Leu Pro Val Thr Ile Met Ser Gly Leu Val Phe 1 5 10 15 His Ser
Gln Pro Ile Asn Asp Xaa 20 24 12 PRT Chimpanzee Immunodeficiency
virus VARIANT (1) modified site 24 Xaa Leu Trp Gly Cys Ser Gly Lys
Ala Val Cys Xaa 1 5 10 25 12 PRT Simian immunodeficiency virus
VARIANT (1) modified site 25 Xaa Ser Trp Gly Cys Ala Trp Lys Gln
Val Cys Xaa 1 5 10 26 12 PRT Simian immunodeficiency virus VARIANT
(1) modified site 26 Xaa Gln Trp Gly Cys Ser Trp Ala Gln Val Cys
Xaa 1 5 10 27 24 PRT Human T-cell lymphotropic virus VARIANT (1)
modified site 27 Xaa Val Leu Tyr Ser Pro Asn Val Ser Val Pro Ser
Ser Ser Ser Thr 1 5 10 15 Leu Leu Tyr Pro Ser Leu Ala Xaa 20 28 23
PRT Human T-cell lymphotropic virus VARIANT (1) modified site 28
Xaa Tyr Thr Cys Ile Val Cys Ile Asp Arg Ala Ser Leu Ser Thr Trp 1 5
10 15 His Val Leu Tyr Ser Pro Xaa 20 29 24 PRT Human T-cell
lymphotropic virus VARIANT (1) modified site 29 Xaa Asn Ser Leu Ile
Leu Pro Pro Phe Ser Leu Ser Pro Val Pro Thr 1 5 10 15 Leu Gly Ser
Arg Ser Arg Arg Xaa 20 30 38 PRT Human T-cell lymphotropic virus
VARIANT (1) modified site 30 Xaa Asp Ala Pro Gly Tyr Asp Pro Ile
Trp Phe Leu Asn Thr Glu Pro 1 5 10 15 Ser Gln Leu Pro Pro Thr Ala
Pro Pro Leu Leu Pro His Ser Asn Leu 20 25 30 Asp His Ile Leu Glu
Xaa 35 31 33 PRT Human T-cell lymphotropic virus VARIANT (1)
modified site 31 Xaa Gln Tyr Ala Ala Gln Asn Arg Arg Gly Leu Asp
Leu Leu Phe Trp 1 5 10 15 Glu Gln Gly Gly Leu Cys Lys Ala Leu Gln
Glu Gln Cys Arg Phe Pro 20 25 30 Xaa 32 33 PRT Human T-cell
lymphotropic virus VARIANT (1) modified site 32 Xaa Pro Pro Pro Pro
Ser Ser Pro Thr His Asp Pro Pro Asp Ser Asp 1 5 10 15 Pro Gln Ile
Pro Pro Pro Tyr Val Glu Pro Thr Ala Pro Gln Val Leu 20 25 30 Xaa 33
20 PRT Human T-cell lymphotropic virus VARIANT (1) modified site 33
Xaa Lys Lys Pro Asn Arg Gln Gly Leu Gly Tyr Tyr Ser Pro Ser Tyr 1 5
10 15 Asn Asp Pro Xaa 20 34 38 PRT Human T-cell lymphotropic virus
VARIANT (1) modified site 34 Xaa Asp Ala Pro Gly Tyr Asp Pro Leu
Trp Phe Ile Thr Ser Glu Pro 1 5 10 15 Thr Gln Pro Pro Pro Thr Ser
Pro Pro Leu Val His Asp Ser Asp Leu 20 25 30 Glu His Val Leu Thr
Xaa 35 35 40 PRT Human T-cell lymphotropic virus VARIANT (1)
modified site 35 Xaa Tyr Ser Cys Met Val Cys Val Asp Arg Ser Ser
Leu Ser Ser Trp 1 5 10 15 His Val Leu Tyr Thr Pro Asn Ile Ser Ile
Pro Gln Gln Thr Ser Ser 20 25 30 Arg Thr Ile Leu Phe Pro Ser Xaa 35
40 36 32 PRT Human T-cell lymphotropic virus VARIANT (1) modified
site 36 Xaa Pro Thr Thr Thr Pro Pro Pro Pro Pro Pro Pro Ser Pro Glu
Ala 1 5 10 15 His Val Pro Pro Pro Tyr Val Glu Pro Thr Thr Thr Gln
Cys Phe Xaa 20 25 30 37 22 PRT Hepatitis C virus VARIANT (1)
modified site 37 Xaa Met Ser Thr Ile Pro Lys Pro Gln Arg Lys Thr
Lys Arg Asn Thr 1 5 10 15 Asn Arg Arg Pro Gln Xaa 20 38 22 PRT
Hepatitis C virus VARIANT (1) modified site 38 Xaa Pro Gln Arg Lys
Thr Lys Arg Asn Thr Asn Arg Arg Pro Gln Asp 1 5 10 15 Val Lys Phe
Pro Gly Xaa 20 39 13 PRT Hepatitis C virus VARIANT (1) modified
site 39 Xaa Gln Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg Xaa 1 5 10
40 22 PRT Hepatitis C virus VARIANT (1) modified site 40 Xaa Arg
Asn Thr Asn Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly 1 5 10 15
Gly Gln Ile Val Gly Xaa 20 41 22 PRT Hepatitis C virus VARIANT (1)
modified site 41 Xaa Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg
Ala Thr Arg Lys 1 5 10 15 Thr Ser Glu Arg Ser Xaa 20 42 22 PRT
Hepatitis C virus VARIANT (1) modified site 42 Xaa Val Gly Gly Val
Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly 1 5 10 15 Val Arg Ala
Thr Arg Xaa 20 43 22 PRT Hepatitis C virus VARIANT (1) modified
site 43 Xaa Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg
Gln 1 5 10 15 Pro Ile Pro Lys Val Xaa 20 44 22 PRT Hepatitis C
virus VARIANT (1) modified site 44 Xaa Arg Ser Gln Pro Arg Gly Arg
Arg Gln Pro Ile Pro Lys Val Arg 1 5 10 15 Arg Pro Glu Gly Arg Xaa
20 45 22 PRT Hepatitis C virus VARIANT (1) modified site 45 Xaa Arg
Arg Gln Pro Ile Pro Lys Val Arg Arg Pro Glu Gly Arg Thr 1 5 10 15
Trp Ala Gln Pro Gly Xaa 20 46 22 PRT Hepatitis C virus VARIANT (1)
modified site 46 Xaa Gly Arg Thr Trp Ala Gln Pro Gly Tyr Pro Trp
Pro Leu Tyr Gly 1 5 10 15 Asn Glu Gly Cys Gly Xaa 20 47 32 PRT
Hepatitis C virus VARIANT (1) modified site 47 Xaa Met Ser Thr Ile
Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn Arg 1 5 10 15 Arg Pro Gln
Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly Xaa 20 25 30 48 42
PRT Hepatitis C virus VARIANT (1) modified site 48 Xaa Gly Gly Val
Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val 1 5 10 15 Arg Arg
Ala Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg 20 25 30
Arg Gln Pro Ile Pro Lys Val Arg Arg Xaa 35 40 49 22 PRT Hepatitis C
virus VARIANT (1) modified site 49 Xaa Leu Ser Gly Lys Pro Ala Ile
Ile Pro Asp Arg Glu Val Leu Tyr 1 5 10 15 Arg Glu Phe Asp Glu Xaa
20 50 22 PRT Hepatitis C virus VARIANT (1) modified site 50 Xaa Ile
Ile Pro Asp Arg Glu Val Leu Tyr Arg Glu Phe Asp Glu Met 1 5 10 15
Glu Glu Cys Ser Gln Xaa 20 51 22 PRT Hepatitis C virus VARIANT (1)
modified site 51 Xaa Val Leu Tyr Arg Glu Phe Asp Glu Met Glu Glu
Cys Ser Gln His 1 5 10 15 Leu Pro Tyr Ile Glu Xaa 20 52 22 PRT
Hepatitis C virus VARIANT (1) modified site 52 Xaa Asp Glu Met Glu
Glu Cys Ser Gln His Leu Pro Tyr Ile Glu Gln 1 5 10 15 Gly Met Met
Leu Ala Xaa 20 53 22 PRT Hepatitis C virus VARIANT (1) modified
site 53 Xaa Ser Gln His Leu Pro Tyr Ile Glu Gln Gly Met Met Leu Ala
Glu 1 5 10 15 Gln Phe Lys Gln Lys Xaa 20 54 22 PRT Hepatitis C
virus VARIANT (1) modified site 54 Xaa Ile Glu Gln Gly Met Met Leu
Ala Glu Gln Phe Lys Gln Lys Ala 1 5 10 15 Leu Gly Leu Leu Gln Xaa
20 55 22 PRT Hepatitis C virus VARIANT (1) modified site 55 Xaa Leu
Ala Glu Gln Phe Lys Gln Lys Ala Leu Gly Leu Leu Gln Thr 1 5 10 15
Ala Ser Arg Gln Ala Xaa 20 56 22 PRT Hepatitis C virus VARIANT (1)
modified site 56 Xaa Gln Lys Ala Leu Gly Leu Leu Gln Thr Ala Ser
Arg Gln Ala Glu 1 5 10 15 Val Ile Ala Pro Ala Xaa 20 57 33 PRT
Hepatitis C virus VARIANT (1) modified site 57 Xaa Ser Gln His Leu
Pro Tyr Ile Glu Gln Glu Met Leu Ala Glu Gln 1 5 10 15 Phe Lys Gln
Lys Ala Leu Gly Leu Leu Gln Thr Ala Ser Arg Gln Ala 20 25 30 Xaa 58
22 PRT Hepatitis C virus VARIANT (1) modified site 58 Xaa Gly Glu
Gly Ala Val Gln Trp Met Asn Arg Leu Ile Ala Phe Ala 1 5 10 15 Ser
Arg Gly Asn His Xaa 20 59 22 PRT Hepatitis C virus VARIANT (1)
modified site 59 Xaa Glu Asp Glu Arg Glu Ile Ser Val Pro Ala Glu
Ile Leu Arg Lys 1 5 10 15 Ser Arg Arg Phe Ala Xaa 20 60 22 PRT
Hepatitis C virus VARIANT (1) modified site 60 Xaa Leu Arg Lys Ser
Arg Arg Phe Ala Gln Ala Leu Pro Val Trp Ala 1 5 10 15 Arg Pro Asp
Tyr Asn Xaa 20 61 22 PRT Hepatitis C virus VARIANT (1) modified
site 61 Xaa Val Trp Ala Arg Pro Asp Tyr Asn Pro Pro Leu Val Glu Thr
Trp 1 5 10 15 Lys Lys Pro Asp Tyr Xaa 20 62 22 PRT Hepatitis C
virus VARIANT (1) modified site 62 Xaa Glu Thr Trp Lys Lys Pro Asp
Tyr Glu Pro Pro Val Val His Gly 1 5 10 15 Cys Pro Leu Pro Pro Xaa
20 63 22 PRT Hepatitis C virus VARIANT (1) modified site 63 Xaa Val
His Gly Cys Pro Leu Pro Pro Pro Lys Ser Pro Pro Val Pro 1 5 10 15
Pro Pro Arg Lys Lys Xaa 20 64 39 PRT Hepatitis C virus VARIANT (1)
modified site 64 Xaa Glu Asp Glu Arg Glu Ile Ser Val Pro Ala Glu
Ile Leu Arg Lys 1 5 10 15 Ser Arg Lys Ser Arg Arg Phe Ala Gln Ala
Leu Pro Val Trp Ala Arg 20 25 30 Pro Asp Tyr Asp Tyr Asn Xaa 35 65
36 PRT Hepatitis C virus VARIANT (1) modified site 65 Xaa Gly Glu
Thr Tyr Thr Ser Gly Gly Ala Ala Ser His Thr Thr Ser 1 5 10 15 Thr
Leu Ala Ser Leu Phe Ser Pro Gly Ala Ser Gln Arg Ile Gln Leu 20 25
30 Val Asn Thr Xaa 35 66 24 PRT Hepatitis C virus VARIANT (1)
modified site 66 Xaa Gly Glu Thr Tyr Thr Ser Gly Gly Ala Ala Ser
His Thr Thr Ser 1 5 10 15 Thr Leu Ala Ser Leu Phe Ser Xaa 20 67 26
PRT Hepatitis C virus VARIANT (1) modified site 67 Xaa Ser His Thr
Thr Ser Thr Leu Ala Ser Leu Phe Ser Pro Gly Ala 1 5 10 15 Ser Gln
Arg Ile Gln Leu Val Asn Thr Xaa 20 25 68 36 PRT Hepatitis C virus
VARIANT (1) modified site 68 Xaa Gly His Thr Arg Val Ser Gly Gly
Ala Ala Ala Ser Asp Thr Arg 1 5 10 15 Gly Leu Val Ser Leu Phe Ser
Pro Gly Ser Ala Gln Lys Ile Gln Leu 20 25 30 Val Asn Thr Xaa 35 69
24 PRT Hepatitis C virus VARIANT (1) modified site 69 Xaa Gly His
Thr Arg Val Ser Gly Gly Ala Ala Ala Ser Asp Thr Arg 1 5 10 15 Gly
Leu Val Ser Leu Phe Ser Xaa 20 70 26 PRT Hepatitis C virus VARIANT
(1) modified site 70 Xaa Ala Ser Asp Thr Arg Gly Leu Val Ser Leu
Phe Ser Pro Gly Ser 1 5 10 15 Ala Gln Lys Ile Gln Leu Val Asn Thr
Xaa 20 25 71 36 PRT Hepatitis C virus VARIANT (1) modified site 71
Xaa Gly His Thr Arg Val Thr Gly Gly Val Gln Gly His Val Thr Cys 1 5
10 15 Thr Leu Thr Ser Leu Phe Arg Pro Gly Ala Ser Gln Lys Ile Gln
Leu 20 25 30 Val Asn Thr Xaa 35 72 24 PRT Hepatitis C virus VARIANT
(1) modified site 72 Xaa Gly His Thr Arg Val Thr Gly Gly Val Gln
Gly His Val Thr Cys 1 5 10 15 Thr Leu Thr Ser Leu Phe Arg Xaa 20 73
26 PRT Hepatitis C virus VARIANT (1) modified site 73 Xaa Gly His
Val Thr Cys Thr Leu Thr Ser Leu Phe Arg Pro Gly Ala 1 5 10 15 Ser
Gln Lys Ile Gln Leu Val Asn Thr Xaa 20 25 74 36 PRT Hepatitis C
virus VARIANT (1) modified site 74 Xaa Gly His Thr His Val Thr Gly
Gly Arg Val Ala Ser Ser Thr Gln 1 5 10 15 Ser Leu Val Ser Trp Leu
Ser Gln Gly Pro Ser Gln Lys Ile Gln Leu 20 25 30 Val Asn Thr Xaa 35
75 24 PRT Hepatitis C virus
VARIANT (1) modified site 75 Xaa Gly His Thr His Val Thr Gly Gly
Arg Val Ala Ser Ser Thr Gln 1 5 10 15 Ser Leu Val Ser Trp Leu Ser
Xaa 20 76 26 PRT Hepatitis C virus VARIANT (1) modified site 76 Xaa
Ala Ser Ser Thr Gln Ser Leu Val Ser Trp Leu Ser Gln Gly Pro 1 5 10
15 Ser Gln Lys Ile Gln Leu Val Asn Thr Xaa 20 25 77 36 PRT
Hepatitis C virus VARIANT (1) modified site 77 Xaa Gly Asp Thr His
Val Thr Gly Gly Ala Gln Ala Lys Thr Thr Asn 1 5 10 15 Arg Leu Val
Ser Met Phe Ala Ser Gly Pro Ser Gln Lys Ile Gln Leu 20 25 30 Ile
Asn Thr Xaa 35 78 24 PRT Hepatitis C virus VARIANT (1) modified
site 78 Xaa Gly Asp Thr His Val Thr Gly Gly Ala Gln Ala Lys Thr Thr
Asn 1 5 10 15 Arg Leu Val Ser Met Phe Ala Xaa 20 79 26 PRT
Hepatitis C virus VARIANT (1) modified site 79 Xaa Ala Lys Thr Thr
Asn Arg Leu Val Ser Met Phe Ala Ser Gly Pro 1 5 10 15 Ser Gln Lys
Ile Gln Leu Ile Asn Thr Xaa 20 25 80 36 PRT Hepatitis C virus
VARIANT (1) modified site 80 Xaa Ala Glu Thr Tyr Thr Ser Gly Gly
Asn Ala Gly His Thr Met Thr 1 5 10 15 Gly Ile Val Arg Phe Phe Ala
Pro Gly Pro Lys Gln Asn Val His Leu 20 25 30 Ile Asn Thr Xaa 35 81
24 PRT Hepatitis C virus VARIANT (1) modified site 81 Xaa Ala Glu
Thr Tyr Thr Ser Gly Gly Asn Ala Gly His Thr Met Thr 1 5 10 15 Gly
Ile Val Arg Phe Phe Ala Xaa 20 82 26 PRT Hepatitis C virus VARIANT
(1) modified site 82 Xaa Gly His Thr Met Thr Gly Ile Val Arg Phe
Phe Ala Pro Gly Pro 1 5 10 15 Lys Gln Asn Val His Leu Ile Asn Thr
Xaa 20 25 83 36 PRT Hepatitis C virus VARIANT (1) modified site 83
Xaa Ala Glu Thr Ile Val Ser Gly Gly Gln Ala Ala Arg Ala Met Ser 1 5
10 15 Gly Leu Val Ser Leu Phe Thr Pro Gly Ala Lys Gln Asn Ile Gln
Leu 20 25 30 Ile Asn Thr Xaa 35 84 24 PRT Hepatitis C virus VARIANT
(1) modified site 84 Xaa Ala Glu Thr Ile Val Ser Gly Gly Gln Ala
Ala Arg Ala Met Ser 1 5 10 15 Gly Leu Val Ser Leu Phe Thr Xaa 20 85
26 PRT Hepatitis C virus VARIANT (1) modified site 85 Xaa Ala Arg
Ala Met Ser Gly Leu Val Ser Leu Phe Thr Pro Gly Ala 1 5 10 15 Lys
Gln Asn Ile Gln Leu Ile Asn Thr Xaa 20 25 86 36 PRT Hepatitis C
virus VARIANT (1) modified site 86 Xaa Ala Glu Thr Tyr Thr Thr Gly
Gly Ser Thr Ala Arg Thr Thr Gln 1 5 10 15 Gly Leu Val Ser Leu Phe
Ser Arg Gly Ala Lys Gln Asp Ile Gln Leu 20 25 30 Ile Asn Thr Xaa 35
87 24 PRT Hepatitis C virus VARIANT (1) modified site 87 Xaa Ala
Glu Thr Tyr Thr Thr Gly Gly Ser Thr Ala Arg Thr Thr Gln 1 5 10 15
Gly Leu Val Ser Leu Phe Ser Xaa 20 88 26 PRT Hepatitis C virus
VARIANT (1) modified site 88 Xaa Ala Arg Thr Thr Gln Gly Leu Val
Ser Leu Phe Ser Arg Gly Ala 1 5 10 15 Lys Gln Asp Ile Gln Leu Ile
Asn Thr Xaa 20 25 89 36 PRT Hepatitis C virus VARIANT (1) modified
site 89 Xaa Ala Gln Thr His Thr Val Gly Gly Ser Thr Ala His Asn Ala
Arg 1 5 10 15 Thr Leu Thr Gly Met Phe Ser Leu Gly Ala Arg Gln Lys
Ile Gln Leu 20 25 30 Ile Asn Thr Xaa 35 90 24 PRT Hepatitis C virus
VARIANT (1) modified site 90 Xaa Ala Gln Thr His Thr Val Gly Gly
Ser Thr Ala His Asn Ala Arg 1 5 10 15 Thr Leu Thr Gly Met Phe Ser
Xaa 20 91 26 PRT Hepatitis C virus VARIANT (1) modified site 91 Xaa
Ala His Asn Ala Arg Thr Leu Thr Gly Met Phe Ser Leu Gly Ala 1 5 10
15 Arg Gln Lys Ile Gln Leu Ile Asn Thr Xaa 20 25 92 22 PRT
Hepatitis C virus VARIANT (1) modified site 92 Xaa Val Asn Gln Arg
Ala Val Val Ala Pro Asp Lys Glu Val Leu Tyr 1 5 10 15 Glu Ala Phe
Asp Glu Xaa 20 93 22 PRT Hepatitis C virus VARIANT (1) modified
site 93 Xaa Val Ala Pro Asp Lys Glu Val Leu Tyr Glu Ala Phe Asp Glu
Met 1 5 10 15 Glu Glu Cys Ala Ser Xaa 20 94 22 PRT Hepatitis C
virus VARIANT (1) modified site 94 Xaa Asp Glu Met Glu Glu Cys Ala
Ser Arg Ala Ala Leu Ile Glu Glu 1 5 10 15 Gly Gln Arg Ile Ala Xaa
20 95 22 PRT Hepatitis C virus VARIANT (1) modified site 95 Xaa Ala
Ser Arg Ala Ala Leu Ile Glu Glu Gly Gln Arg Ile Ala Glu 1 5 10 15
Met Leu Lys Ser Lys Xaa 20 96 22 PRT Hepatitis C virus VARIANT (1)
modified site 96 Xaa Ile Glu Glu Gly Gln Arg Ile Ala Glu Met Leu
Lys Ser Lys Ile 1 5 10 15 Gln Gly Leu Leu Gln Xaa 20 97 22 PRT
Hepatitis C virus VARIANT (1) modified site 97 Xaa Ile Ala Glu Met
Leu Lys Ser Lys Ile Gln Gly Leu Leu Gln Gln 1 5 10 15 Ala Ser Lys
Gln Ala Xaa 20 98 22 PRT Hepatitis C virus VARIANT (1) modified
site 98 Xaa Ser Lys Ile Gln Gly Leu Leu Gln Gln Ala Ser Lys Gln Ala
Gln 1 5 10 15 Asp Ile Gln Pro Ala Xaa 20 99 22 PRT Hepatitis C
virus VARIANT (1) modified site 99 Xaa Arg Ser Asp Leu Glu Pro Ser
Ile Pro Ser Glu Tyr Met Leu Pro 1 5 10 15 Lys Lys Arg Phe Pro Xaa
20 100 22 PRT Hepatitis C virus VARIANT (1) modified site 100 Xaa
Met Leu Pro Lys Lys Arg Phe Pro Pro Ala Leu Pro Ala Trp Ala 1 5 10
15 Arg Pro Asp Tyr Asn Xaa 20 101 22 PRT Hepatitis C virus VARIANT
(1) modified site 101 Xaa Ala Trp Ala Arg Pro Asp Tyr Asn Pro Pro
Leu Val Glu Ser Trp 1 5 10 15 Lys Arg Pro Asp Tyr Xaa 20 102 22 PRT
Hepatitis C virus VARIANT (1) modified site 102 Xaa Glu Ser Trp Lys
Arg Pro Asp Tyr Gln Pro Ala Thr Val Ala Gly 1 5 10 15 Cys Ala Leu
Pro Pro Xaa 20 103 22 PRT Hepatitis C virus VARIANT (1) modified
site 103 Xaa Val Ala Gly Cys Ala Leu Pro Pro Pro Lys Lys Thr Pro
Thr Pro 1 5 10 15 Pro Pro Arg Arg Arg Xaa 20 104 22 PRT Hepatitis C
virus VARIANT (1) modified site 104 Xaa Leu Gly Gly Lys Pro Ala Ile
Val Pro Asp Lys Glu Val Leu Tyr 1 5 10 15 Gln Gln Tyr Asp Glu Xaa
20 105 22 PRT Hepatitis C virus VARIANT (1) modified site 105 Xaa
Ser Gln Ala Ala Pro Tyr Ile Glu Gln Ala Gln Val Ile Ala His 1 5 10
15 Gln Phe Lys Glu Lys Xaa 20 106 24 PRT Hepatitis C virus VARIANT
(1) modified site 106 Xaa Ile Ala His Gln His Gln Phe Lys Glu Lys
Val Leu Gly Leu Leu 1 5 10 15 Gln Arg Ala Thr Gln Gln Gln Xaa 20
107 33 PRT Hepatitis C virus VARIANT (1) modified site 107 Xaa Ile
Pro Asp Arg Glu Val Leu Tyr Arg Gly Gly Lys Lys Pro Asp 1 5 10 15
Tyr Glu Pro Pro Val Gly Gly Arg Arg Pro Gln Asp Val Lys Phe Pro 20
25 30 Xaa 108 33 PRT Hepatitis C virus VARIANT (1) modified site
108 Xaa Trp Ala Arg Pro Asp Tyr Asn Pro Pro Gly Gly Gln Phe Lys Gln
1 5 10 15 Lys Ala Leu Gly Leu Gly Ser Gly Val Tyr Leu Leu Pro Arg
Arg Gly 20 25 30 Xaa 109 33 PRT Hepatitis C virus VARIANT (1)
modified site 109 Xaa Arg Gly Arg Arg Gln Pro Ile Pro Lys Gly Gly
Ser Gln His Leu 1 5 10 15 Pro Tyr Ile Glu Gln Ser Gly Pro Val Val
His Gly Cys Pro Leu Pro 20 25 30 Xaa 110 12 PRT Human
immunodeficiency virus VARIANT (1) modified site Ac 110 Xaa Ile Trp
Gly Cys Ser Gly Lys Leu Ile Cys Xaa 1 5 10 111 15 PRT Human
immunodeficiency virus VARIANT (1) modified site Bio 111 Xaa Gly
Gly Gly Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys Xaa 1 5 10 15 112
12 PRT Human immunodeficiency virus VARIANT (1) modified site Ac
112 Xaa Ser Trp Gly Cys Ala Phe Arg Gln Val Cys Xaa 1 5 10 113 15
PRT Human immunodeficiency virus VARIANT (1) modified site Bio 113
Xaa Gly Gly Gly Ser Trp Gly Cys Ala Phe Arg Gln Val Cys Xaa 1 5 10
15 114 25 PRT Human immunodeficiency virus VARIANT (1) modified
site Ac 114 Xaa Tyr Asn Lys Arg Lys Arg Ile His Ile Gly Pro Gly Arg
Ala Phe 1 5 10 15 Tyr Thr Thr Lys Asn Ile Ile Gly Xaa 20 25 115 26
PRT Human immunodeficiency virus VARIANT (1) modified site Bio 115
Xaa Gly Gly Tyr Asn Lys Arg Lys Arg Ile His Ile Gly Pro Gly Arg 1 5
10 15 Ala Phe Thr Thr Lys Asn Ile Ile Gly Xaa 20 25 116 20 PRT
Hepatitis C virus 116 Ser Gln His Leu Pro Tyr Ile Glu Gln Gly Met
Met Leu Ala Glu Gln 1 5 10 15 Phe Lys Gln Lys 20 117 20 PRT
Hepatitis C virus 117 Leu Arg Lys Ser Arg Arg Phe Ala Gln Ala Leu
Pro Val Trp Ala Arg 1 5 10 15 Pro Asp Tyr Asn 20 118 19 PRT
Hepatitis C virus 118 Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn Arg
Arg Pro Gln Asp Val 1 5 10 15 Lys Phe Gly 119 20 PRT Hepatitis C
virus 119 Arg Asn Thr Asn Arg Arg Pro Gln Asp Val Lys Phe Pro Gly
Gly Gly 1 5 10 15 Gln Ile Val Gly 20 120 20 PRT Hepatitis C virus
120 Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro
1 5 10 15 Ile Pro Lys Val 20 121 20 PRT Hepatitis C virus 121 Ile
Ile Pro Asp Arg Glu Val Leu Tyr Arg Glu Phe Asp Glu Met Glu 1 5 10
15 Glu Cys Ser Gln 20 122 20 PRT Hepatitis C virus 122 Glu Thr Trp
Lys Lys Pro Asp Tyr Glu Pro Pro Val Val His Gly Cys 1 5 10 15 Pro
Leu Pro Pro 20 123 21 PRT Hepatitis C virus VARIANT (1) modified
site NH2 123 Xaa Met Ser Thr Ile Pro Lys Pro Gln Arg Lys Thr Lys
Arg Asn Thr 1 5 10 15 Asn Arg Pro Gln Xaa 20 124 24 PRT Hepatitis C
virus VARIANT (1) modified site NH2 124 Xaa Met Ser Thr Ile Pro Lys
Pro Gln Arg Lys Thr Lys Arg Asn Thr 1 5 10 15 Asn Arg Pro Gln Gly
Gly Xaa Xaa 20 125 34 PRT Hepatitis C virus 125 Gly Glu Thr Tyr Thr
Ser Gly Gly Ala Ala Ser His Thr Thr Ser Thr 1 5 10 15 Leu Ala Ser
Leu Phe Ser Pro Gly Ala Ser Gln Arg Ile Gln Leu Val 20 25 30 Asn
Thr 126 34 PRT Hepatitis C virus 126 Gly His Thr Arg Val Ser Gly
Gly Ala Ala Ala Ser Asp Thr Arg Gly 1 5 10 15 Leu Val Ser Leu Phe
Ser Pro Gly Ser Ala Gln Lys Ile Gln Leu Val 20 25 30 Asn Thr 127 34
PRT Hepatitis C virus 127 Gly His Thr Arg Val Thr Gly Gly Val Gln
Gly His Val Thr Cys Thr 1 5 10 15 Leu Thr Ser Leu Phe Arg Pro Gly
Ala Ser Gln Lys Ile Gln Leu Val 20 25 30 Asn Thr 128 34 PRT
Hepatitis C virus 128 Gly His Thr His Val Thr Gly Gly Arg Val Ala
Ser Ser Thr Gln Ser 1 5 10 15 Leu Val Ser Trp Leu Ser Gln Gly Pro
Ser Gln Lys Ile Gln Leu Val 20 25 30 Asn Thr 129 34 PRT Hepatitis C
virus 129 Gly Asp Thr His Val Thr Gly Gly Ala Gln Ala Lys Thr Thr
Asn Arg 1 5 10 15 Leu Val Ser Met Phe Ala Ser Gly Pro Ser Gln Lys
Ile Gln Leu Ile 20 25 30 Asn Thr 130 34 PRT Hepatitis C virus 130
Ala Glu Thr Tyr Thr Ser Gly Gly Asn Ala Gly His Thr Met Thr Gly 1 5
10 15 Ile Val Arg Phe Phe Ala Pro Gly Pro Lys Gln Asn Val His Leu
Ile 20 25 30 Asn Thr 131 34 PRT Hepatitis C virus 131 Ala Glu Thr
Ile Val Ser Gly Gly Gln Ala Ala Arg Ala Met Ser Gly 1 5 10 15 Leu
Val Ser Leu Phe Thr Pro Gly Ala Lys Gln Asn Ile Gln Leu Ile 20 25
30 Asn Thr 132 34 PRT Hepatitis C virus 132 Ala Glu Thr Tyr Thr Thr
Gly Gly Ser Thr Ala Arg Thr Thr Gln Gly 1 5 10 15 Leu Val Ser Leu
Phe Ser Arg Gly Ala Lys Gln Asp Ile Gln Leu Ile 20 25 30 Asn Thr
133 20 PRT Hepatitis C virus 133 Met Ser Thr Ile Pro Lys Pro Gln
Arg Lys Thr Lys Arg Asn Thr Asn 1 5 10 15 Arg Arg Pro Gln 20 134 15
PRT Hepatitis C virus 134 Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn
Thr Asn Arg Arg Pro 1 5 10 15 135 20 PRT Hepatitis C virus 135 Arg
Asn Thr Asn Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly 1 5 10
15 Gln Ile Val Gly 20 136 32 PRT Hepatitis C virus 136 Met Ser Thr
Ile Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn 1 5 10 15 Arg
Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly 20 25
30 137 20 PRT Hepatitis C virus 137 Val Gly Gly Val Tyr Leu Leu Pro
Arg Arg Gly Pro Arg Leu Gly Val 1 5 10 15 Arg Ala Thr Arg 20 138 20
PRT Hepatitis C virus 138 Leu Pro Arg Arg Gly Pro Arg Leu Gly Val
Arg Ala Thr Arg Lys Thr 1 5 10 15 Ser Glu Arg Ser 20 139 20 PRT
Hepatitis C virus 139 Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg
Gly Arg Arg Gln Pro 1 5 10 15 Ile Pro Glu Val 20 140 20 PRT
Hepatitis C virus 140 Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro Ile
Pro Glu Val Arg Arg 1 5 10 15 Pro Glu Gly Arg 20 141 39 PRT
Hepatitis C virus 141 Gly Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro
Arg Leu Gly Val Arg 1 5 10 15 Ala Thr Arg Lys Thr Ser Glu Arg Ser
Gln Pro Arg Gly Arg Arg Gln 20 25 30 Pro Ile Pro Lys Val Arg Arg 35
142 20 PRT Hepatitis C virus 142 Ser Gln His Leu Pro Tyr Ile Glu
Gln Gly Met Met Leu Ala Glu Gln 1 5 10 15 Phe Lys Gln Lys 20 143 20
PRT Hepatitis C virus 143 Leu Ala Glu Gln Phe Lys Gln Lys Ala Leu
Gly Leu Leu Gln Thr Ala 1 5 10 15 Ser Arg Gln Ala 20 144 32 PRT
Hepatitis C virus 144 Ser Gln His Leu Pro Tyr Ile Glu Gln Gly Met
Met Leu Ala Glu Gln 1 5 10 15 Phe Lys Gln Lys Ala Leu Gly Leu Leu
Gln Thr Ala Ser Arg Gln Ala 20 25 30 145 20 PRT Hepatitis C virus
145 Glu Asp Glu Arg Glu Ile Ser Val Pro Ala Glu Ile Leu Arg Lys Ser
1 5 10 15 Arg Arg Phe Ala 20 146 20 PRT Hepatitis C virus 146 Leu
Arg Lys Ser Arg Arg Phe Ala Gln Ala Leu Pro Val Trp Ala Arg 1 5 10
15 Pro Asp Tyr Asn 20 147 32 PRT Hepatitis C virus 147 Glu Asp Glu
Arg Glu Ile Ser Val Pro Ala Glu Ile Leu Arg Lys Ser 1 5 10 15 Arg
Arg Phe Ala Gln Ala Leu Pro Val Trp Ala Arg Pro Asp Tyr Asn 20 25
30 148 20 PRT Hepatitis C virus 148 Leu Ser Gly Lys Pro Ala Ile Ile
Pro Asp Arg Glu Val Leu Tyr Arg 1 5 10 15 Glu Phe Asp Glu 20 149 20
PRT Hepatitis C virus 149 Val Asn Gln Arg Ala Val Val Ala Pro Asp
Lys Glu Val Leu Tyr Glu 1 5 10 15 Ala Phe Asp Glu 20 150 20 PRT
Hepatitis C virus 150 Ser Gln His Leu Pro Tyr Ile Glu Gln Gly Met
Met Leu Ala Glu Gln 1 5 10 15 Phe Lys Gln Lys 20 151 20 PRT
Hepatitis C virus 151 Ala Ser Arg Ala Ala Leu Ile Glu Glu Gly Gln
Arg Ile Ala Glu Met 1 5 10 15 Leu Lys Ser Lys 20 152 20 PRT
Hepatitis C virus 152 Leu Ala Glu Gln Phe Lys Gln Lys Ala Leu Gly
Leu Leu Gln Thr Ala 1 5 10 15 Ser Arg Gln Ala 20 153 20
PRT Hepatitis C virus 153 Ile Ala Glu Met Leu Lys Ser Lys Ile Gln
Gly Leu Leu Gln Gln Ala 1 5 10 15 Ser Lys Gln Ala 20 154 23 PRT
Human immunodeficiency virus 154 Asn Asn Thr Arg Lys Ser Ile His
Ile Gly Pro Gly Arg Ala Phe Tyr 1 5 10 15 Thr Thr Gly Glu Ile Ile
Gly 20 155 23 PRT Human immunodeficiency virus 155 Asn Asn Thr Arg
Lys Ser Ile Tyr Ile Gly Pro Gly Arg Ala Phe His 1 5 10 15 Thr Thr
Gly Arg Ile Ile Gly 20 156 23 PRT Human immunodeficiency virus 156
Asn Asn Thr Thr Arg Ser Ile His Ile Gly Pro Gly Arg Ala Phe Tyr 1 5
10 15 Ala Thr Gly Asp Ile Ile Gly 20 157 23 PRT Human
immunodeficiency virus 157 Tyr Asn Lys Arg Lys Arg Ile His Ile Gly
Pro Gly Arg Ala Phe Tyr 1 5 10 15 Thr Thr Lys Asn Ile Ile Gly 20
158 23 PRT Human immunodeficiency virus 158 Asn Asn Thr Arg Lys Ser
Ile Thr Lys Gly Pro Gly Arg Val Ile Tyr 1 5 10 15 Ala Thr Gly Gln
Ile Ile Gly 20 159 22 PRT Human immunodeficiency virus 159 Asn Asn
Thr Arg Arg Gly Ile His Phe Gly Pro Gly Gln Ala Leu Tyr 1 5 10 15
Thr Thr Gly Ile Val Gly 20 160 24 PRT Human immunodeficiency virus
160 Asn Asn Thr Arg Lys Ser Ile Arg Ile Gln Arg Gly Pro Gly Arg Ala
1 5 10 15 Phe Val Thr Ile Gly Lys Ile Gly 20 161 22 PRT Human
immunodeficiency virus 161 Gln Asn Thr Arg Gln Arg Thr Pro Ile Gly
Leu Gly Gln Ser Leu Tyr 1 5 10 15 Thr Thr Arg Ser Arg Ser 20 162 21
PRT Human immunodeficiency virus 162 Gln Ile Asp Ile Gln Glu Met
Arg Ile Gly Pro Met Ala Trp Tyr Ser 1 5 10 15 Met Gly Ile Gly Gly
20 163 23 PRT Human immunodeficiency virus 163 Asn Asn Thr Arg Arg
Gly Ile His Met Gly Trp Gly Arg Thr Phe Tyr 1 5 10 15 Ala Thr Gly
Glu Ile Ile Gly 20 164 22 PRT Human immunodeficiency virus 164 Asn
Lys Thr Val Val Pro Ile Thr Leu Met Ser Gly Leu Val Phe His 1 5 10
15 Ser Gln Pro Ile Asn Lys 20 165 22 PRT Human immunodeficiency
virus 165 Asn Lys Thr Val Leu Pro Val Thr Ile Met Ser Gly Leu Val
Phe His 1 5 10 15 Ser Gln Pro Ile Asn Asp 20 166 25 PRT Human
T-cell lymphotropic virus VARIANT (1) modified site Bio 166 Xaa Gly
Gly Val Leu Tyr Ser Pro Asn Val Ser Val Pro Ser Ser Ser 1 5 10 15
Ser Thr Leu Leu Tyr Pro Ser Leu Ala 20 25 167 24 PRT Human T-cell
lymphotropic virus VARIANT (1) modified site Bio 167 Xaa Gly Gly
Tyr Thr Cys Ile Val Cys Ile Asp Arg Ala Ser Leu Ser 1 5 10 15 Thr
Trp His Val Leu Tyr Ser Pro 20 168 25 PRT Human T-cell lymphotropic
virus VARIANT (1) modified site 168 Xaa Gly Gly Asn Ser Leu Ile Leu
Pro Pro Phe Ser Leu Ser Pro Val 1 5 10 15 Pro Thr Leu Gly Ser Arg
Ser Arg Arg 20 25 169 39 PRT Human T-cell lymphotropic virus
VARIANT (1) modified site Bio 169 Xaa Gly Gly Asp Ala Pro Gly Tyr
Asp Pro Ile Trp Phe Leu Asn Thr 1 5 10 15 Glu Pro Ser Gln Leu Pro
Pro Thr Ala Pro Pro Leu Leu Pro His Ser 20 25 30 Asn Leu Asp His
Ile Leu Glu 35 170 34 PRT Human T-cell lymphotropic virus VARIANT
(1) modified site Bio 170 Xaa Gly Gly Gln Tyr Ala Ala Gln Asn Arg
Arg Gly Leu Asp Leu Leu 1 5 10 15 Phe Trp Glu Gln Gly Gly Leu Cys
Lys Ala Leu Gln Glu Gln Cys Arg 20 25 30 Phe Pro 171 34 PRT Human
T-cell lymphotropic virus VARIANT (1) modified site Bio 171 Xaa Gly
Gly Pro Pro Pro Pro Ser Ser Pro Thr His Asp Pro Pro Asp 1 5 10 15
Ser Asp Pro Gln Ile Pro Pro Pro Tyr Val Glu Pro Thr Ala Pro Gln 20
25 30 Val Leu 172 21 PRT Human T-cell lymphotropic virus VARIANT
(1) modified site Bio 172 Xaa Gly Gly Lys Lys Pro Asn Arg Gln Gly
Leu Gly Tyr Tyr Ser Pro 1 5 10 15 Ser Tyr Asn Asp Pro 20 173 39 PRT
Human T-cell lymphotropic virus VARIANT (1) modified site 173 Xaa
Gly Gly Asp Ala Pro Gly Tyr Asp Pro Leu Trp Phe Ile Thr Ser 1 5 10
15 Glu Pro Thr Gln Pro Pro Pro Thr Ser Pro Pro Leu Val His Asp Ser
20 25 30 Asp Leu Glu His Val Leu Thr 35 174 41 PRT Human T-cell
lymphotropic virus VARIANT (1) modified site Bio 174 Xaa Gly Gly
Tyr Ser Cys Met Val Cys Val Asp Arg Ser Ser Leu Ser 1 5 10 15 Ser
Trp His Val Leu Tyr Thr Pro Asn Ile Ser Ile Pro Gln Gln Thr 20 25
30 Ser Ser Arg Thr Ile Leu Phe Pro Ser 35 40 175 33 PRT Human
T-cell lymphotropic virus VARIANT (1) modified site Bio 175 Xaa Gly
Gly Pro Thr Thr Thr Pro Pro Pro Pro Pro Pro Pro Ser Pro 1 5 10 15
Glu Ala His Val Pro Pro Pro Tyr Val Glu Pro Thr Thr Thr Gln Cys 20
25 30 Phe 176 14 PRT Human immunodeficiency virus VARIANT (1)
modified site Bio 176 Xaa Gly Gly Gly Ile Trp Gly Cys Ser Gly Lys
Leu Ile Cys 1 5 10 177 13 PRT Human immunodeficiency virus VARIANT
(13) modified site Lys (Bio) 177 Ile Trp Gly Cys Ser Gly Lys Leu
Ile Cys Gly Gly Xaa 1 5 10 178 9 PRT Hepatitis C virus 178 Met Ser
Thr Ile Pro Lys Pro Gln Arg 1 5 179 9 PRT Hepatitis C virus 179 Ser
Thr Ile Pro Lys Pro Gln Arg Lys 1 5 180 9 PRT Hepatitis C virus 180
Thr Ile Pro Lys Pro Gln Arg Lys Thr 1 5 181 9 PRT Hepatitis C virus
181 Ile Pro Lys Pro Gln Arg Lys Thr Lys 1 5 182 9 PRT Hepatitis C
virus 182 Pro Lys Pro Gln Arg Lys Thr Lys Arg 1 5 183 9 PRT
Hepatitis C virus 183 Lys Pro Gln Arg Lys Thr Lys Arg Asn 1 5 184 9
PRT Hepatitis C virus 184 Pro Gln Arg Lys Thr Lys Arg Asn Thr 1 5
185 9 PRT Hepatitis C virus 185 Gln Arg Lys Thr Lys Arg Asn Thr Asn
1 5 186 9 PRT Hepatitis C virus 186 Arg Lys Thr Lys Arg Asn Thr Asn
Arg 1 5 187 9 PRT Hepatitis C virus 187 Lys Thr Lys Arg Asn Thr Asn
Arg Arg 1 5 188 9 PRT Hepatitis C virus 188 Thr Lys Arg Asn Thr Asn
Arg Arg Pro 1 5 189 9 PRT Hepatitis C virus 189 Lys Arg Asn Thr Asn
Arg Arg Pro Gln 1 5 190 9 PRT Hepatitis C virus 190 Arg Asn Thr Asn
Arg Arg Pro Gln Asp 1 5 191 9 PRT Hepatitis C virus 191 Asn Thr Asn
Arg Arg Pro Gln Asp Val 1 5 192 9 PRT Hepatitis C virus 192 Thr Asn
Arg Arg Pro Gln Asp Val Lys 1 5 193 9 PRT Hepatitis C virus 193 Asn
Arg Arg Pro Gln Asp Val Lys Phe 1 5 194 9 PRT Hepatitis C virus 194
Arg Arg Pro Gln Asp Val Lys Phe Pro 1 5 195 9 PRT Hepatitis C virus
195 Arg Pro Gln Asp Val Lys Phe Pro Gly 1 5 196 9 PRT Hepatitis C
virus 196 Pro Gln Asp Val Lys Phe Pro Gly Gly 1 5 197 9 PRT
Hepatitis C virus 197 Gln Asp Val Lys Phe Pro Gly Gly Gly 1 5 198 9
PRT Hepatitis C virus 198 Asp Val Lys Phe Pro Gly Gly Gly Gln 1 5
199 9 PRT Hepatitis C virus 199 Val Lys Phe Pro Gly Gly Gly Gln Ile
1 5 200 9 PRT Hepatitis C virus 200 Lys Phe Pro Gly Gly Gly Gln Ile
Val 1 5 201 9 PRT Hepatitis C virus 201 Phe Pro Gly Gly Gly Gln Ile
Val Gly 1 5 202 9 PRT Hepatitis C virus 202 Pro Gly Gly Gly Gln Ile
Val Gly Gly 1 5 203 9 PRT Hepatitis C virus 203 Gly Gly Gly Gln Ile
Val Gly Gly Val 1 5 204 9 PRT Hepatitis C virus 204 Gly Gly Gln Ile
Val Gly Gly Val Tyr 1 5 205 9 PRT Hepatitis C virus 205 Gly Gln Ile
Val Gly Gly Val Tyr Leu 1 5 206 9 PRT Hepatitis C virus 206 Gln Ile
Val Gly Gly Val Tyr Leu Leu 1 5 207 9 PRT Hepatitis C virus 207 Ile
Val Gly Gly Val Tyr Leu Leu Pro 1 5 208 9 PRT Hepatitis C virus 208
Val Gly Gly Val Tyr Leu Leu Pro Arg 1 5 209 9 PRT Hepatitis C virus
209 Gly Gly Val Tyr Leu Leu Pro Arg Arg 1 5 210 9 PRT Hepatitis C
virus 210 Gly Val Tyr Leu Leu Pro Arg Arg Gly 1 5 211 9 PRT
Hepatitis C virus 211 Val Tyr Leu Leu Pro Arg Arg Gly Pro 1 5 212 9
PRT Hepatitis C virus 212 Tyr Leu Leu Pro Arg Arg Gly Pro Arg 1 5
213 9 PRT Hepatitis C virus 213 Leu Leu Pro Arg Arg Gly Pro Arg Leu
1 5 214 9 PRT Hepatitis C virus 214 Leu Pro Arg Arg Gly Pro Arg Leu
Gly 1 5 215 9 PRT Hepatitis C virus 215 Pro Arg Arg Gly Pro Arg Leu
Gly Val 1 5 216 9 PRT Hepatitis C virus 216 Arg Arg Gly Pro Arg Leu
Gly Val Arg 1 5 217 9 PRT Hepatitis C virus 217 Arg Gly Pro Arg Leu
Gly Val Arg Ala 1 5 218 9 PRT Hepatitis C virus 218 Gly Pro Arg Leu
Gly Val Arg Ala Thr 1 5 219 9 PRT Hepatitis C virus 219 Pro Arg Leu
Gly Val Arg Ala Thr Arg 1 5 220 9 PRT Hepatitis C virus 220 Arg Leu
Gly Val Arg Ala Thr Arg Lys 1 5 221 9 PRT Hepatitis C virus 221 Leu
Gly Val Arg Ala Thr Arg Lys Thr 1 5 222 9 PRT Hepatitis C virus 222
Gly Val Arg Ala Thr Arg Lys Thr Ser 1 5 223 9 PRT Hepatitis C virus
223 Val Arg Ala Thr Arg Lys Thr Ser Glu 1 5 224 9 PRT Hepatitis C
virus 224 Arg Ala Thr Arg Lys Thr Ser Glu Arg 1 5 225 9 PRT
Hepatitis C virus 225 Ala Thr Arg Lys Thr Ser Glu Arg Ser 1 5 226 9
PRT Hepatitis C virus 226 Thr Arg Lys Thr Ser Glu Arg Ser Gln 1 5
227 9 PRT Hepatitis C virus 227 Arg Lys Thr Ser Glu Arg Ser Gln Pro
1 5 228 9 PRT Hepatitis C virus 228 Lys Thr Ser Glu Arg Ser Gln Pro
Arg 1 5 229 9 PRT Hepatitis C virus 229 Thr Ser Glu Arg Ser Gln Pro
Arg Gly 1 5 230 9 PRT Hepatitis C virus 230 Ser Glu Arg Ser Gln Pro
Arg Gly Arg 1 5 231 9 PRT Hepatitis C virus 231 Glu Arg Ser Gln Pro
Arg Gly Arg Arg 1 5 232 9 PRT Hepatitis C virus 232 Arg Ser Gln Pro
Arg Gly Arg Arg Gln 1 5 233 9 PRT Hepatitis C virus 233 Ser Gln Pro
Arg Gly Arg Arg Gln Pro 1 5 234 9 PRT Hepatitis C virus 234 Gln Pro
Arg Gly Arg Arg Gln Pro Ile 1 5 235 9 PRT Hepatitis C virus 235 Pro
Arg Gly Arg Arg Gln Pro Ile Pro 1 5 236 9 PRT Hepatitis C virus 236
Arg Gly Arg Arg Gln Pro Ile Pro Lys 1 5 237 9 PRT Hepatitis C virus
237 Gly Arg Arg Gln Pro Ile Pro Lys Val 1 5 238 9 PRT Hepatitis C
virus 238 Arg Arg Gln Pro Ile Pro Lys Val Arg 1 5 239 9 PRT
Hepatitis C virus 239 Arg Gln Pro Ile Pro Lys Val Arg Arg 1 5 240 9
PRT Hepatitis C virus 240 Gln Pro Ile Pro Lys Val Arg Arg Pro 1 5
241 9 PRT Hepatitis C virus 241 Pro Ile Pro Lys Val Arg Arg Pro Glu
1 5 242 9 PRT Hepatitis C virus 242 Ile Pro Lys Val Arg Arg Pro Glu
Gly 1 5 243 9 PRT Hepatitis C virus 243 Pro Lys Val Arg Arg Pro Glu
Gly Arg 1 5 244 9 PRT Hepatitis C virus 244 Lys Val Arg Arg Pro Glu
Gly Arg Thr 1 5 245 9 PRT Hepatitis C virus 245 Val Arg Arg Pro Glu
Gly Arg Thr Trp 1 5 246 9 PRT Hepatitis C virus 246 Arg Arg Pro Glu
Gly Arg Thr Trp Ala 1 5 247 9 PRT Hepatitis C virus 247 Arg Pro Glu
Gly Arg Thr Trp Ala Gln 1 5 248 9 PRT Hepatitis C virus 248 Pro Glu
Gly Arg Thr Trp Ala Gln Pro 1 5 249 9 PRT Hepatitis C virus 249 Glu
Gly Arg Thr Trp Ala Gln Pro Gly 1 5 250 9 PRT Hepatitis C virus 250
Gly Arg Thr Trp Ala Gln Pro Gly Tyr 1 5 251 9 PRT Hepatitis C virus
251 Arg Thr Trp Ala Gln Pro Gly Tyr Pro 1 5 252 9 PRT Hepatitis C
virus 252 Thr Trp Ala Gln Pro Gly Tyr Pro Trp 1 5 253 9 PRT
Hepatitis C virus 253 Trp Ala Gln Pro Gly Tyr Pro Trp Pro 1 5 254 9
PRT Hepatitis C virus 254 Ala Gln Pro Gly Tyr Pro Trp Pro Leu 1 5
255 9 PRT Hepatitis C virus 255 Gln Pro Gly Tyr Pro Trp Pro Leu Tyr
1 5 256 9 PRT Hepatitis C virus 256 Pro Gly Tyr Pro Trp Pro Leu Tyr
Gly 1 5 257 9 PRT Hepatitis C virus 257 Gly Tyr Pro Trp Pro Leu Tyr
Gly Asn 1 5 258 9 PRT Hepatitis C virus 258 Leu Ser Gly Lys Pro Ala
Ile Ile Pro 1 5 259 9 PRT Hepatitis C virus 259 Ser Gly Lys Pro Ala
Ile Ile Pro Asp 1 5 260 9 PRT Hepatitis C virus 260 Gly Lys Pro Ala
Ile Ile Pro Asp Arg 1 5 261 9 PRT Hepatitis C virus 261 Lys Pro Ala
Ile Ile Pro Asp Arg Glu 1 5 262 9 PRT Hepatitis C virus 262 Pro Ala
Ile Ile Pro Asp Arg Glu Val 1 5 263 9 PRT Hepatitis C virus 263 Ala
Ile Ile Pro Asp Arg Glu Val Leu 1 5 264 9 PRT Hepatitis C virus 264
Ile Ile Pro Asp Arg Glu Val Leu Tyr 1 5 265 9 PRT Hepatitis C virus
265 Ile Pro Asp Arg Glu Val Leu Tyr Arg 1 5 266 9 PRT Hepatitis C
virus 266 Pro Asp Arg Glu Val Leu Tyr Arg Glu 1 5 267 9 PRT
Hepatitis C virus 267 Asp Arg Glu Val Leu Tyr Arg Glu Phe 1 5 268 9
PRT Hepatitis C virus 268 Arg Glu Val Leu Tyr Arg Glu Phe Asp 1 5
269 9 PRT Hepatitis C virus 269 Glu Val Leu Tyr Arg Glu Phe Asp Glu
1 5 270 9 PRT Hepatitis C virus 270 Val Leu Tyr Arg Glu Phe Asp Glu
Met 1 5 271 9 PRT Hepatitis C virus 271 Leu Tyr Arg Glu Phe Asp Glu
Met Glu 1 5 272 9 PRT Hepatitis C virus 272 Tyr Arg Glu Phe Asp Glu
Met Glu Glu 1 5 273 9 PRT Hepatitis C virus 273 Arg Glu Phe Asp Glu
Met Glu Glu Cys 1 5 274 9 PRT Hepatitis C virus 274 Glu Phe Asp Glu
Met Glu Glu Cys Ser 1 5 275 9 PRT Hepatitis C virus 275 Phe Asp Glu
Met Glu Glu Cys Ser Gln 1 5 276 9 PRT Hepatitis C virus 276 Asp Glu
Met Glu Glu Cys Ser Gln His 1 5 277 9 PRT Hepatitis C virus 277 Glu
Met Glu Glu Cys Ser Gln His Leu 1 5 278 9 PRT Hepatitis C virus 278
Met Glu Glu Cys Ser Gln His Leu Pro 1 5 279 9 PRT Hepatitis C virus
279 Glu Glu Cys Ser Gln His Leu Pro Tyr 1 5 280 9 PRT Hepatitis C
virus 280 Glu Cys Ser Gln His Leu Pro Tyr Ile 1 5 281 9 PRT
Hepatitis C virus 281 Cys Ser Gln His Leu Pro Tyr Ile Glu 1 5 282 9
PRT Hepatitis C virus 282 Ser Gln His Leu Pro Tyr Ile Glu Gln 1 5
283 9 PRT Hepatitis C virus 283 Gln His Leu Pro Tyr Ile Glu Gln Gly
1 5 284 9 PRT Hepatitis C virus 284 His Leu Pro Tyr Ile Glu Gln Gly
Met 1 5 285 9 PRT Hepatitis C virus 285 Leu Pro Tyr Ile Glu Gln Gly
Met Met 1 5 286 9 PRT Hepatitis C virus 286 Pro Tyr Ile Glu Gln Gly
Met Met Leu 1 5 287 9 PRT Hepatitis C virus 287 Tyr Ile Glu Gln Gly
Met Met Leu Ala 1 5 288 9 PRT Hepatitis C virus 288 Ile Glu Gln Gly
Met Met Leu Ala Glu 1 5 289 9 PRT Hepatitis C virus 289 Glu Gln Gly
Met Met Leu Ala Glu Gln 1 5 290 9 PRT Hepatitis C virus 290 Gln Gly
Met Met Leu Ala Glu Gln Phe 1 5 291 9 PRT Hepatitis C virus 291 Gly
Met Met Leu Ala Glu Gln Phe Lys 1 5 292 9 PRT Hepatitis C virus 292
Met Met Leu Ala Glu Gln Phe Lys Gln 1 5 293 9 PRT Hepatitis C virus
293 Met Leu Ala Glu Gln Phe Lys Gln Lys 1 5 294 9 PRT Hepatitis C
virus 294 Leu Ala Glu Gln Phe Lys Gln Lys Ala 1 5 295 9 PRT
Hepatitis C virus 295 Ala Glu Gln Phe Lys Gln Lys Ala Leu 1 5 296 9
PRT Hepatitis C virus 296 Glu Gln Phe Lys Gln Lys Ala Leu Gly 1 5
297 9 PRT Hepatitis C virus 297 Gln Phe Lys Gln Lys Ala Leu Gly Leu
1 5 298 9 PRT Hepatitis C virus 298 Phe Lys Gln Lys Ala Leu Gly Leu
Leu 1 5 299 9 PRT Hepatitis C virus 299 Lys Gln Lys Ala Leu Gly Leu
Leu Gln 1 5 300 9 PRT Hepatitis C virus 300 Gln Lys Ala Leu Gly Leu
Leu Gln Thr 1 5 301 9 PRT Hepatitis C virus 301 Lys Ala Leu Gly Leu
Leu Gln Thr Ala 1 5 302 9 PRT Hepatitis C virus 302 Ala Leu Gly Leu
Leu Gln Thr Ala Ser 1 5 303 9 PRT Hepatitis C virus 303 Leu Gly Leu
Leu Gln Thr Ala Ser Arg 1 5 304 9 PRT Hepatitis C virus 304 Gly Leu
Leu Gln Thr Ala Ser Arg Gln 1 5 305 9 PRT Hepatitis C virus 305 Leu
Leu Gln Thr Ala Ser Arg Gln Ala 1 5 306 9 PRT Hepatitis C virus 306
Leu Gln Thr Ala Ser Arg Gln Ala Glu 1 5 307 9 PRT Hepatitis C virus
307 Gln Thr Ala Ser Arg Gln Ala Glu Val 1 5 308 9 PRT Hepatitis C
virus 308 Thr Ala Ser Arg Gln Ala Glu Val Ile 1 5 309 9 PRT
Hepatitis C virus 309 Ala Ser Arg Gln Ala Glu Val Ile Ala 1 5 310 9
PRT Hepatitis C virus 310 Ser Arg Gln Ala Glu Val Ile Ala Pro 1 5
311 9 PRT Hepatitis C virus 311 Arg Gln Ala Glu Val Ile Ala Pro Ala
1 5 312 9 PRT Hepatitis C virus 312 Gln Ala Glu Val Ile Ala Pro Ala
Val 1 5 313 9 PRT Hepatitis C virus 313
Ala Glu Val Ile Ala Pro Ala Val Gln 1 5 314 9 PRT Hepatitis C virus
314 Glu Val Ile Ala Pro Ala Val Gln Thr 1 5 315 9 PRT Hepatitis C
virus 315 Val Ile Ala Pro Ala Val Gln Thr Asn 1 5 316 9 PRT
Hepatitis C virus 316 Ile Ala Pro Ala Val Gln Thr Asn Trp 1 5 317 9
PRT Hepatitis C virus 317 Ala Pro Ala Val Gln Thr Asn Trp Gln 1 5
318 9 PRT Hepatitis C virus 318 Gly Asn Ile Thr Arg Tyr Glu Ser Glu
1 5 319 9 PRT Hepatitis C virus 319 Asn Ile Thr Arg Tyr Glu Ser Glu
Asn 1 5 320 9 PRT Hepatitis C virus 320 Ile Thr Arg Tyr Glu Ser Glu
Asn Lys 1 5 321 9 PRT Hepatitis C virus 321 Thr Arg Tyr Glu Ser Glu
Asn Lys Val 1 5 322 9 PRT Hepatitis C virus 322 Arg Tyr Glu Ser Glu
Asn Lys Val Val 1 5 323 9 PRT Hepatitis C virus 323 Tyr Glu Ser Glu
Asn Lys Val Val Ile 1 5 324 9 PRT Hepatitis C virus 324 Glu Ser Glu
Asn Lys Val Val Ile Leu 1 5 325 9 PRT Hepatitis C virus 325 Ser Glu
Asn Lys Val Val Ile Leu Asp 1 5 326 9 PRT Hepatitis C virus 326 Glu
Asn Lys Val Val Ile Leu Asp Ser 1 5 327 9 PRT Hepatitis C virus 327
Asn Lys Val Val Ile Leu Asp Ser Phe 1 5 328 9 PRT Hepatitis C virus
328 Lys Val Val Ile Leu Asp Ser Phe Asp 1 5 329 9 PRT Hepatitis C
virus 329 Val Val Ile Leu Asp Ser Phe Asp Pro 1 5 330 9 PRT
Hepatitis C virus 330 Val Ile Leu Asp Ser Phe Asp Pro Leu 1 5 331 9
PRT Hepatitis C virus 331 Ile Leu Asp Ser Phe Asp Pro Leu Val 1 5
332 9 PRT Hepatitis C virus 332 Leu Asp Ser Phe Asp Pro Leu Val Ala
1 5 333 9 PRT Hepatitis C virus 333 Asp Ser Phe Asp Pro Leu Val Ala
Glu 1 5 334 9 PRT Hepatitis C virus 334 Ser Phe Asp Pro Leu Val Ala
Glu Glu 1 5 335 9 PRT Hepatitis C virus 335 Phe Asp Pro Leu Val Ala
Glu Glu Asp 1 5 336 9 PRT Hepatitis C virus 336 Asp Pro Leu Val Ala
Glu Glu Asp Glu 1 5 337 9 PRT Hepatitis C virus 337 Pro Leu Val Ala
Glu Glu Asp Glu Arg 1 5 338 9 PRT Hepatitis C virus 338 Leu Val Ala
Glu Glu Asp Glu Arg Glu 1 5 339 9 PRT Hepatitis C virus 339 Val Ala
Glu Glu Asp Glu Arg Glu Ile 1 5 340 9 PRT Hepatitis C virus 340 Ala
Glu Glu Asp Glu Arg Glu Ile Ser 1 5 341 9 PRT Hepatitis C virus 341
Glu Glu Asp Glu Arg Glu Ile Ser Val 1 5 342 9 PRT Hepatitis C virus
342 Glu Asp Glu Arg Glu Ile Ser Val Pro 1 5 343 9 PRT Hepatitis C
virus 343 Asp Glu Arg Glu Ile Ser Val Pro Ala 1 5 344 9 PRT
Hepatitis C virus 344 Glu Arg Glu Ile Ser Val Pro Ala Glu 1 5 345 9
PRT Hepatitis C virus 345 Arg Glu Ile Ser Val Pro Ala Glu Ile 1 5
346 9 PRT Hepatitis C virus 346 Glu Ile Ser Val Pro Ala Glu Ile Leu
1 5 347 9 PRT Hepatitis C virus 347 Ile Ser Val Pro Ala Glu Ile Leu
Arg 1 5 348 9 PRT Hepatitis C virus 348 Ser Val Pro Ala Glu Ile Leu
Arg Lys 1 5 349 9 PRT Hepatitis C virus 349 Val Pro Ala Glu Ile Leu
Arg Lys Ser 1 5 350 9 PRT Hepatitis C virus 350 Pro Ala Glu Ile Leu
Arg Lys Ser Arg 1 5 351 9 PRT Hepatitis C virus 351 Ala Glu Ile Leu
Arg Lys Ser Arg Arg 1 5 352 9 PRT Hepatitis C virus 352 Glu Ile Leu
Arg Lys Ser Arg Arg Phe 1 5 353 9 PRT Hepatitis C virus 353 Ile Leu
Arg Lys Ser Arg Arg Phe Ala 1 5 354 9 PRT Hepatitis C virus 354 Leu
Arg Lys Ser Arg Arg Phe Ala Gln 1 5 355 9 PRT Hepatitis C virus 355
Arg Lys Ser Arg Arg Phe Ala Gln Ala 1 5 356 9 PRT Hepatitis C virus
356 Lys Ser Arg Arg Phe Ala Gln Ala Leu 1 5 357 9 PRT Hepatitis C
virus 357 Ser Arg Arg Phe Ala Gln Ala Leu Pro 1 5 358 9 PRT
Hepatitis C virus 358 Arg Arg Phe Ala Gln Ala Leu Pro Val 1 5 359 9
PRT Hepatitis C virus 359 Arg Phe Ala Gln Ala Leu Pro Val Trp 1 5
360 9 PRT Hepatitis C virus 360 Phe Ala Gln Ala Leu Pro Val Trp Ala
1 5 361 9 PRT Hepatitis C virus 361 Ala Gln Ala Leu Pro Val Trp Ala
Arg 1 5 362 9 PRT Hepatitis C virus 362 Gln Ala Leu Pro Val Trp Ala
Arg Pro 1 5 363 9 PRT Hepatitis C virus 363 Ala Leu Pro Val Trp Ala
Arg Pro Asp 1 5 364 9 PRT Hepatitis C virus 364 Leu Pro Val Trp Ala
Arg Pro Asp Tyr 1 5 365 9 PRT Hepatitis C virus 365 Pro Val Trp Ala
Arg Pro Asp Tyr Asn 1 5 366 9 PRT Hepatitis C virus 366 Val Trp Ala
Arg Pro Asp Tyr Asn Pro 1 5 367 9 PRT Hepatitis C virus 367 Trp Ala
Arg Pro Asp Tyr Asn Pro Pro 1 5 368 9 PRT Hepatitis C virus 368 Ala
Arg Pro Asp Tyr Asn Pro Pro Leu 1 5 369 9 PRT Hepatitis C virus 369
Arg Pro Asp Tyr Asn Pro Pro Leu Val 1 5 370 9 PRT Hepatitis C virus
370 Pro Asp Tyr Asn Pro Pro Leu Val Glu 1 5 371 9 PRT Hepatitis C
virus 371 Asp Tyr Asn Pro Pro Leu Val Glu Thr 1 5 372 9 PRT
Hepatitis C virus 372 Tyr Asn Pro Pro Leu Val Glu Thr Trp 1 5 373 9
PRT Hepatitis C virus 373 Asn Pro Pro Leu Val Glu Thr Trp Lys 1 5
374 9 PRT Hepatitis C virus 374 Pro Pro Leu Val Glu Thr Trp Lys Lys
1 5 375 9 PRT Hepatitis C virus 375 Pro Leu Val Glu Thr Trp Lys Lys
Pro 1 5 376 9 PRT Hepatitis C virus 376 Leu Val Glu Thr Trp Lys Lys
Pro Asp 1 5 377 9 PRT Hepatitis C virus 377 Val Glu Thr Trp Lys Lys
Pro Asp Tyr 1 5 378 9 PRT Hepatitis C virus 378 Glu Thr Trp Lys Lys
Pro Asp Tyr Glu 1 5 379 9 PRT Hepatitis C virus 379 Thr Trp Lys Lys
Pro Asp Tyr Glu Pro 1 5 380 9 PRT Hepatitis C virus 380 Trp Lys Lys
Pro Asp Tyr Glu Pro Pro 1 5 381 9 PRT Hepatitis C virus 381 Lys Lys
Pro Asp Tyr Glu Pro Pro Val 1 5 382 9 PRT Hepatitis C virus 382 Lys
Pro Asp Tyr Glu Pro Pro Val Val 1 5 383 9 PRT Hepatitis C virus 383
Lys Pro Asp Tyr Glu Pro Pro Val Val 1 5 384 9 PRT Hepatitis C virus
384 Asp Tyr Glu Pro Pro Val Val His Gly 1 5 385 9 PRT Hepatitis C
virus 385 Tyr Glu Pro Pro Val Val His Gly Cys 1 5 386 9 PRT
Hepatitis C virus 386 Glu Pro Pro Val Val His Gly Cys Pro 1 5 387 9
PRT Hepatitis C virus 387 Pro Pro Val Val His Gly Cys Pro Leu 1 5
388 9 PRT Hepatitis C virus 388 Pro Val Val His Gly Cys Pro Leu Pro
1 5 389 9 PRT Hepatitis C virus 389 Val Val His Gly Cys Pro Leu Pro
Pro 1 5 390 9 PRT Hepatitis C virus 390 Val His Gly Cys Pro Leu Pro
Pro Pro 1 5 391 9 PRT Hepatitis C virus 391 His Gly Cys Pro Leu Pro
Pro Pro Lys 1 5 392 9 PRT Hepatitis C virus 392 Gly Cys Pro Leu Pro
Pro Pro Lys Ser 1 5 393 9 PRT Hepatitis C virus 393 Cys Pro Leu Pro
Pro Pro Lys Ser Pro 1 5 394 9 PRT Hepatitis C virus 394 Pro Leu Pro
Pro Pro Lys Ser Pro Pro 1 5 395 9 PRT Hepatitis C virus 395 Leu Pro
Pro Pro Lys Ser Pro Pro Val 1 5 396 9 PRT Hepatitis C virus 396 Pro
Pro Pro Lys Ser Pro Pro Val Pro 1 5 397 9 PRT Hepatitis C virus 397
Pro Pro Lys Ser Pro Pro Val Pro Pro 1 5 398 9 PRT Hepatitis C virus
398 Pro Lys Ser Pro Pro Val Pro Pro Pro 1 5 399 9 PRT Hepatitis C
virus 399 Lys Ser Pro Pro Val Pro Pro Pro Arg 1 5 400 9 PRT
Hepatitis C virus 400 Ser Pro Pro Val Pro Pro Pro Arg Lys 1 5 401 9
PRT Hepatitis C virus 401 Pro Pro Val Pro Pro Pro Arg Lys Lys 1 5
402 34 PRT Hepatitis C virus VARIANT (1) Xaa = modified site when
present, represents an amino acid, amino group, or chemically
modified amino terminus 402 Xaa Met Ser Thr Ile Pro Lys Pro Gln Arg
Lys Thr Lys Arg Asn Thr 1 5 10 15 Asn Arg Arg Pro Gln Asp Val Lys
Phe Pro Gly Gly Gly Gln Ile Val 20 25 30 Gly Xaa 403 41 PRT
Hepatitis C virus VARIANT (1) Xaa = modified site when present,
represents an amino acid, amino group, or chemically modified amino
terminus 403 Xaa Gly Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg
Leu Gly Val 1 5 10 15 Arg Ala Thr Arg Lys Thr Ser Glu Arg Ser Gln
Pro Arg Gly Arg Arg 20 25 30 Gln Pro Ile Pro Lys Val Arg Arg Xaa 35
40 404 34 PRT Hepatitis C virus VARIANT (1) Xaa = modified site
when present, represents an amino acid, amino group, or chemically
modified amino terminus 404 Xaa Ser Gln His Leu Pro Tyr Ile Glu Gln
Gly Met Met Leu Ala Glu 1 5 10 15 Gln Phe Lys Gln Lys Ala Leu Gly
Leu Leu Gln Thr Ala Ser Arg Gln 20 25 30 Ala Xaa 405 34 PRT
Hepatitis C virus VARIANT (1) Xaa = modified site when present,
represents an amino acid, amino group, or chemically modified amino
terminuss 405 Xaa Glu Asp Glu Arg Glu Ile Ser Val Pro Ala Glu Ile
Leu Arg Lys 1 5 10 15 Ser Arg Arg Phe Ala Gln Ala Leu Pro Val Trp
Ala Arg Pro Asp Tyr 20 25 30 Asn Xaa 406 22 PRT Hepatitis C virus
406 Gly Glu Thr Tyr Thr Ser Gly Gly Ala Ala Ser His Thr Thr Ser Thr
1 5 10 15 Leu Ala Ser Leu Phe Ser 20 407 24 PRT Hepatitis C virus
407 Ser His Thr Thr Ser Thr Leu Ala Ser Leu Phe Ser Pro Gly Ala Ser
1 5 10 15 Gln Arg Ile Gln Leu Val Asn Thr 20 408 22 PRT Hepatitis C
virus 408 Gly His Thr Arg Val Ser Gly Gly Ala Ala Ala Ser Asp Thr
Arg Gly 1 5 10 15 Leu Val Ser Leu Phe Ser 20 409 24 PRT Hepatitis C
virus 409 Ala Ser Asp Thr Arg Gly Leu Val Ser Leu Phe Ser Pro Gly
Ser Ala 1 5 10 15 Gln Lys Ile Gln Leu Val Asn Thr 20 410 22 PRT
Hepatitis C virus 410 Gly His Thr Arg Val Thr Gly Gly Val Gln Gly
His Val Thr Cys Thr 1 5 10 15 Leu Thr Ser Leu Phe Arg 20 411 24 PRT
Hepatitis C virus 411 Gly His Val Thr Cys Thr Leu Thr Ser Leu Phe
Arg Pro Gly Ala Ser 1 5 10 15 Gln Lys Ile Gln Leu Val Asn Thr 20
412 22 PRT Hepatitis C virus 412 Gly His Thr His Val Thr Gly Gly
Arg Val Ala Ser Ser Thr Gln Ser 1 5 10 15 Leu Val Ser Trp Leu Ser
20 413 24 PRT Hepatitis C virus 413 Ala Ser Ser Thr Gln Ser Leu Val
Ser Trp Leu Ser Gln Gly Pro Ser 1 5 10 15 Gln Lys Ile Gln Leu Val
Asn Thr 20 414 22 PRT Hepatitis C virus 414 Gly Asp Thr His Val Thr
Gly Gly Ala Gln Ala Lys Thr Thr Asn Arg 1 5 10 15 Leu Val Ser Met
Phe Ala 20 415 24 PRT Hepatitis C virus 415 Ala Lys Thr Thr Asn Arg
Leu Val Ser Met Phe Ala Ser Gly Pro Ser 1 5 10 15 Gln Lys Ile Gln
Leu Ile Asn Thr 20 416 22 PRT Hepatitis C virus 416 Ala Glu Thr Tyr
Thr Ser Gly Gly Asn Ala Gly His Thr Met Thr Gly 1 5 10 15 Ile Val
Arg Phe Phe Ala 20 417 24 PRT Hepatitis C virus 417 Gly His Thr Met
Thr Gly Ile Val Arg Phe Phe Ala Pro Gly Pro Lys 1 5 10 15 Gln Asn
Val His Leu Ile Asn Thr 20 418 22 PRT Hepatitis C virus 418 Ala Glu
Thr Ile Val Ser Gly Gly Gln Ala Ala Arg Ala Met Ser Gly 1 5 10 15
Leu Val Ser Leu Phe Thr 20 419 24 PRT Hepatitis C virus 419 Ala Arg
Ala Met Ser Gly Leu Val Ser Leu Phe Thr Pro Gly Ala Lys 1 5 10 15
Gln Asn Ile Gln Leu Ile Asn Thr 20 420 22 PRT Hepatitis C virus 420
Ala Glu Thr Tyr Thr Thr Gly Gly Ser Thr Ala Arg Thr Thr Gln Gly 1 5
10 15 Leu Val Ser Leu Phe Ser 20 421 24 PRT Hepatitis C virus 421
Ala Arg Thr Thr Gln Gly Leu Val Ser Leu Phe Ser Arg Gly Ala Lys 1 5
10 15 Gln Asp Ile Gln Leu Ile Asn Thr 20 422 6 PRT Hepatitis C
virus 422 Pro Gln Arg Lys Thr Lys 1 5 423 7 PRT Hepatitis C virus
423 Lys Thr Lys Arg Asn Thr Asn 1 5 424 7 PRT Hepatitis C virus 424
Pro Gln Asp Val Lys Phe Pro 1 5 425 6 PRT Hepatitis C virus 425 Tyr
Leu Leu Pro Arg Arg 1 5 426 7 PRT Hepatitis C virus 426 Pro Arg Arg
Gly Pro Arg Leu 1 5 427 7 PRT Hepatitis C virus 427 Arg Leu Gly Val
Arg Ala Thr 1 5 428 7 PRT Hepatitis C virus 428 Ser Gln Pro Arg Gly
Arg Arg 1 5 429 7 PRT Hepatitis C virus 429 Arg Arg Gln Pro Ile Pro
Lys 1 5 430 6 PRT Hepatitis C virus 430 Arg Thr Trp Ala Gln Pro 1 5
431 8 PRT Hepatitis C virus 431 Gln Pro Gly Tyr Pro Trp Pro Leu 1 5
432 6 PRT Hepatitis C virus 432 Pro Asp Arg Glu Val Leu 1 5 433 6
PRT Hepatitis C virus 433 His Leu Pro Tyr Ile Glu 1 5 434 8 PRT
Hepatitis C virus 434 Tyr Ile Glu Gln Gly Met Met Leu 1 5 435 7 PRT
Hepatitis C virus 435 Ala Glu Gln Phe Lys Gln Lys 1 5 436 6 PRT
Hepatitis C virus 436 Lys Gln Lys Ala Leu Gly 1 5 437 7 PRT
Hepatitis C virus 437 Leu Gly Leu Leu Gln Thr Ala 1 5 438 7 PRT
Hepatitis C virus 438 Pro Ala Glu Ile Leu Arg Lys 1 5 439 7 PRT
Hepatitis C virus 439 Glu Ile Leu Arg Lys Ser Arg 1 5 440 7 PRT
Hepatitis C virus 440 Gln Ala Leu Pro Val Trp Ala 1 5 441 6 PRT
Hepatitis C virus 441 Pro Asp Tyr Asn Pro Pro 1 5 442 7 PRT
Hepatitis C virus 442 Leu Val Glu Thr Trp Lys Lys 1 5 443 6 PRT
Hepatitis C virus 443 Asp Tyr Glu Pro Pro Val 1 5 444 5 PRT
Hepatitis C virus 444 His Gly Cys Pro Leu 1 5 445 20 PRT Hepatitis
C virus 445 Gly Ala Leu Val Ala Phe Lys Ile Met Ser Gly Glu Val Pro
Ser Thr 1 5 10 15 Glu Asp Leu Val 20 446 20 PRT Hepatitis C virus
446 Val Pro Ser Thr Glu Asp Leu Val Asn Leu Leu Pro Ala Ile Leu Ser
1 5 10 15 Pro Gly Ala Leu 20 447 20 PRT Hepatitis C virus 447 Ala
Ile Leu Ser Pro Gly Ala Leu Val Val Gly Val Val Cys Ala Ala 1 5 10
15 Ile Leu Arg Arg 20 448 20 PRT Hepatitis C virus 448 Val Cys Ala
Ala Ile Leu Arg Arg His Val Gly Pro Gly Glu Gly Ala 1 5 10 15 Val
Gln Trp Met 20 449 20 PRT Hepatitis C virus 449 Gly Glu Gly Ala Val
Gln Trp Met Asn Arg Leu Ile Ala Phe Ala Ser 1 5 10 15 Arg Gly Asn
His 20 450 34 PRT Hepatitis C virus 450 Gly Gly Ile Pro Asp Arg Glu
Val Leu Tyr Arg Gly Gly Lys Lys Pro 1 5 10 15 Asp Thr Tyr Glu Pro
Pro Val Gly Gly Arg Arg Pro Gln Asp Val Lys 20 25 30 Phe Pro 451 33
PRT Hepatitis C virus 451 Gly Gly Trp Ala Arg Pro Asp Tyr Asn Pro
Pro Gly Gly Gln Phe Lys 1 5 10 15 Gln Lys Ala Leu Gly Leu Gly Ser
Gly Val Tyr Leu Leu Pro Arg Arg 20 25 30 Gly 452 33 PRT Hepatitis C
virus 452 Gly Gly Arg Gly Arg Arg Gln Pro Ile Pro Lys Gly Gly Ser
Gln His 1 5 10 15 Leu Pro Tyr Ile Glu Gln Ser Gly Pro Val Val His
Gly Cys Pro Leu 20 25 30 Pro 453 36 PRT Hepatitis C virus VARIANT
(1) Xaa = modified site when present, represents an amino acid,
amino group, or chemically modified amino terminus 453 Ala Gly Glu
Thr Tyr Thr Ser Gly Gly Ala Ala Ser His Thr Thr Ser 1 5 10 15 Thr
Leu Ala Ser Leu Phe Ser Pro Gly Ala Ser Gln Arg Ile Gln Leu 20 25
30 Val Asn Thr Glx 35 454 36 PRT Hepatitis C virus VARIANT (1) Xaa
= modified site when present, represents an amino acid, amino
group, or chemically modified amino terminus 454 Ala Gly His Thr
Arg Val Ser Gly Gly Ala Ala Ala Ser Asp Thr Arg 1 5 10 15 Gly Leu
Val Ser Leu Phe Ser Pro Gly Ser Ala Gln Lys Ile Gln Leu 20 25 30
Val Asn Thr Glx 35 455 36 PRT Hepatitis C virus VARIANT (1) Xaa =
modified site when present, represents an amino acid, amino group,
or chemically modified amino terminus 455 Ala Gly His Thr Arg Val
Thr Gly Gly Val Gln Gly His Val Thr Cys 1 5 10 15 Thr Leu Thr Ser
Leu Phe Arg Pro Gly Ala Ser Gln Lys Ile Gln Leu 20 25 30 Val Asn
Thr Glx 35 456 36 PRT Hepatitis C virus VARIANT (1) Xaa = modified
site when present, represents an amino acid, amino group, or
chemically modified amino terminus 456 Ala Gly His Thr His Val Thr
Gly Gly Arg Val Ala Ser Ser Thr Gln 1 5 10 15 Ser Leu Val Ser Trp
Leu Ser Gln Gly Pro Ser Gln Lys Ile Gln Leu 20 25 30 Val Asn Thr
Glx 35 457 36 PRT Hepatitis C virus
VARIANT (1) Xaa = modified site when present, represents an amino
acid, amino group, or chemically modified amino terminus 457 Ala
Gly Asp Thr His Val Thr Gly Gly Ala Gln Ala Lys Thr Thr Asn 1 5 10
15 Arg Leu Val Ser Met Phe Ala Ser Gly Pro Ser Gln Lys Ile Gln Leu
20 25 30 Ile Asn Thr Glx 35 458 36 PRT Hepatitis C virus VARIANT
(1) Xaa = modified site when present, represents an amino acid,
amino group, or chemically modified amino terminus 458 Ala Ala Glu
Thr Tyr Thr Ser Gly Gly Asn Ala Gly His Thr Met Thr 1 5 10 15 Gly
Ile Val Arg Phe Phe Ala Pro Gly Pro Lys Gln Asn Val His Leu 20 25
30 Ile Asn Thr Glx 35 459 36 PRT Hepatitis C virus VARIANT (1) Xaa
= modified site when present, represents an amino acid, amino
group, or chemically modified amino terminus 459 Ala Ala Glu Thr
Ile Val Ser Gly Gly Gln Ala Ala Arg Ala Met Ser 1 5 10 15 Gly Leu
Val Ser Leu Phe Thr Pro Gly Ala Lys Gln Asn Ile Gln Leu 20 25 30
Ile Asn Thr Glx 35 460 36 PRT Hepatitis C virus VARIANT (1) Xaa =
modified site when present, represents an amino acid, amino group,
or chemically modified amino terminus 460 Ala Ala Glu Thr Tyr Thr
Thr Gly Gly Ser Thr Ala Arg Thr Thr Gln 1 5 10 15 Gly Leu Val Ser
Leu Phe Ser Arg Gly Ala Lys Gln Asp Ile Gln Leu 20 25 30 Ile Asn
Thr Glx 35 461 24 PRT Hepatitis C virus VARIANT (1) Xaa = modified
site when present, represents an amino acid, amino group, or
chemically modified amino terminus 461 Ala Gly Glu Thr Tyr Thr Ser
Gly Gly Ala Ala Ser His Thr Thr Ser 1 5 10 15 Thr Leu Ala Ser Leu
Phe Ser Glx 20 462 26 PRT Hepatitis C virus VARIANT (1) Xaa =
modified site when present, represents an amino acid, amino group,
or chemically modified amino terminus 462 Ala Ser His Thr Thr Ser
Thr Leu Ala Ser Leu Phe Ser Pro Gly Ala 1 5 10 15 Ser Gln Arg Ile
Gln Leu Val Asn Thr Glx 20 25 463 24 PRT Hepatitis C virus VARIANT
(1) Xaa = modified site when present, represents an amino acid,
amino group, or chemically modified amino terminus 463 Ala Gly His
Thr Arg Val Ser Gly Gly Ala Ala Ala Ser Asp Thr Arg 1 5 10 15 Gly
Leu Val Ser Leu Phe Ser Glx 20 464 26 PRT Hepatitis C virus VARIANT
(1) Xaa = modified site when present, represents an amino acid,
amino group, or chemically modified amino terminus 464 Ala Ala Ser
Asp Thr Arg Gly Leu Val Ser Leu Phe Ser Pro Gly Ser 1 5 10 15 Ala
Gln Lys Ile Gln Leu Val Asn Thr Glx 20 25 465 24 PRT Hepatitis C
virus VARIANT (1) Xaa = modified site when present, represents an
amino acid, amino group, or chemically modified amino terminus 465
Ala Gly His Thr Arg Val Thr Gly Gly Val Gln Gly His Val Thr Cys 1 5
10 15 Thr Leu Thr Ser Leu Phe Arg Glx 20 466 26 PRT Hepatitis C
virus VARIANT (1) Xaa = modified site when present, represents an
amino acid, amino group, or chemically modified amino terminus 466
Ala Gly His Val Thr Cys Thr Leu Thr Ser Leu Phe Arg Pro Gly Ala 1 5
10 15 Ser Gln Lys Ile Gln Leu Val Asn Thr Glx 20 25 467 24 PRT
Hepatitis C virus VARIANT (1) Xaa = modified site when present,
represents an amino acid, amino group, or chemically modified amino
terminus 467 Ala Gly His Thr His Val Thr Gly Gly Arg Val Ala Ser
Ser Thr Gln 1 5 10 15 Ser Leu Val Ser Trp Leu Ser Glx 20 468 26 PRT
Hepatitis C virus VARIANT (1) Xaa = modified site when present,
represents an amino acid, amino group, or chemically modified amino
terminus 468 Ala Ala Ser Ser Thr Gln Ser Leu Val Ser Trp Leu Ser
Gln Gly Pro 1 5 10 15 Ser Gln Lys Ile Gln Leu Val Asn Thr Glx 20 25
469 24 PRT Hepatitis C virus VARIANT (1) Xaa = modified site when
present, represents an amino acid, amino group, or chemically
modified amino terminus 469 Ala Gly Asp Thr His Val Thr Gly Gly Ala
Gln Ala Lys Thr Thr Asn 1 5 10 15 Arg Leu Val Ser Met Phe Ala Glx
20 470 26 PRT Hepatitis C virus VARIANT (1) Xaa = modified site
when present, represents an amino acid, amino group, or chemically
modified amino terminus 470 Ala Ala Lys Thr Thr Asn Arg Leu Val Ser
Met Phe Ala Ser Gly Pro 1 5 10 15 Ser Gln Lys Ile Gln Leu Ile Asn
Thr Glx 20 25 471 24 PRT Hepatitis C virus VARIANT (1) Xaa =
modified site when present, represents an amino acid, amino group,
or chemically modified amino terminus 471 Ala Ala Glu Thr Tyr Thr
Ser Gly Gly Asn Ala Gly His Thr Met Thr 1 5 10 15 Gly Ile Val Arg
Phe Phe Ala Glx 20 472 26 PRT Hepatitis C virus VARIANT (1) Xaa =
modified site when present, represents an amino acid, amino group,
or chemically modified amino terminus 472 Ala Gly His Thr Met Thr
Gly Ile Val Arg Phe Phe Ala Pro Gly Pro 1 5 10 15 Lys Gln Asn Val
His Leu Ile Asn Thr Glx 20 25 473 24 PRT Hepatitis C virus VARIANT
(1) Xaa = modified site when present, represents an amino acid,
amino group, or chemically modified amino terminuss 473 Ala Ala Glu
Thr Ile Val Ser Gly Gly Gln Ala Ala Arg Ala Met Ser 1 5 10 15 Gly
Leu Val Ser Leu Phe Thr Glx 20 474 26 PRT Hepatitis C virus VARIANT
(1) Xaa = modified site when present, represents an amino acid,
amino group, or chemically modified amino terminus 474 Ala Ala Arg
Ala Met Ser Gly Leu Val Ser Leu Phe Thr Pro Gly Ala 1 5 10 15 Lys
Gln Asn Ile Gln Leu Ile Asn Thr Glx 20 25 475 24 PRT Hepatitis C
virus VARIANT (1) Xaa = modified site when present, represents an
amino acid, amino group, or chemically modified amino terminus 475
Ala Ala Glu Thr Tyr Thr Thr Gly Gly Ser Thr Ala Arg Thr Thr Gln 1 5
10 15 Gly Leu Val Ser Leu Phe Ser Glx 20 476 26 PRT Hepatitis C
virus VARIANT (1) Xaa = modified site when present, represents an
amino acid, amino group, or chemically modified amino terminus 476
Ala Ala Arg Thr Thr Gln Gly Leu Val Ser Leu Phe Ser Arg Gly Ala 1 5
10 15 Lys Gln Asp Ile Gln Leu Ile Asn Thr Glx 20 25 477 11 PRT
Hepatitis C virus VARIANT (1) Xaa = modified site when present,
represents an amino acid, amino group, or chemically modified amino
terminus 477 Ala Ile Pro Lys Pro Gln Arg Lys Thr Lys Glx 1 5 10 478
11 PRT Hepatitis C virus VARIANT (1) Xaa = modified site when
present, represents an amino acid, amino group, or chemically
modified amino terminus 478 Ala Pro Lys Pro Gln Arg Lys Thr Lys Arg
Glx 1 5 10 479 11 PRT Hepatitis C virus VARIANT (1) Xaa = modified
site when present, represents an amino acid, amino group, or
chemically modified amino terminus 479 Ala Lys Pro Gln Arg Lys Thr
Lys Arg Asn Glx 1 5 10 480 11 PRT Hepatitis C virus VARIANT (1) Xaa
= modified site when present, represents an amino acid, amino
group, or chemically modified amino terminus 480 Ala Pro Gln Arg
Lys Thr Lys Arg Asn Thr Glx 1 5 10 481 11 PRT Hepatitis C virus
VARIANT (1) Xaa = modified site when present, represents an amino
acid, amino group, or chemically modified amino terminus 481 Ala
Gln Arg Lys Thr Lys Arg Asn Thr Asn Glx 1 5 10 482 11 PRT Hepatitis
C virus VARIANT (1) Xaa = modified site when present, represents an
amino acid, amino group, or chemically modified amino terminus 482
Ala Arg Lys Thr Lys Arg Asn Thr Asn Arg Glx 1 5 10 483 11 PRT
Hepatitis C virus VARIANT (1) Xaa = modified site when present,
represents an amino acid, amino group, or chemically modified amino
terminus 483 Ala Lys Thr Lys Arg Asn Thr Asn Arg Arg Glx 1 5 10 484
11 PRT Hepatitis C virus VARIANT (1) Xaa = modified site when
present, represents an amino acid, amino group, or chemically
modified amino terminus 484 Ala Thr Lys Arg Asn Thr Asn Arg Arg Pro
Glx 1 5 10 485 11 PRT Hepatitis C virus VARIANT (1) Xaa = modified
site when present, represents an amino acid, amino group, or
chemically modified amino terminus 485 Ala Arg Arg Pro Gln Asp Val
Lys Phe Pro Glx 1 5 10 486 11 PRT Hepatitis C virus VARIANT (1) Xaa
= modified site when present, represents an amino acid, amino
group, or chemically modified amino terminus 486 Ala Arg Pro Gln
Asp Val Lys Phe Pro Gly Glx 1 5 10 487 11 PRT Hepatitis C virus
VARIANT (1) Xaa = modified site when present, represents an amino
acid, amino group, or chemically modified amino terminus 487 Ala
Pro Gln Asp Val Lys Phe Pro Gly Gly Glx 1 5 10 488 11 PRT Hepatitis
C virus VARIANT (1) Xaa = modified site when present, represents an
amino acid, amino group, or chemically modified amino terminus 488
Ala Gln Asp Val Lys Phe Pro Gly Gly Gly Glx 1 5 10 489 11 PRT
Hepatitis C virus VARIANT (1) Xaa = modified site when present,
represents an amino acid, amino group, or chemically modified amino
terminus 489 Ala Asp Val Lys Phe Pro Gly Gly Gly Gln Glx 1 5 10 490
11 PRT Hepatitis C virus VARIANT (1) Xaa = modified site when
present, represents an amino acid, amino group, or chemically
modified amino terminus 490 Ala Gly Gly Val Tyr Leu Leu Pro Arg Arg
Glx 1 5 10 491 11 PRT Hepatitis C virus VARIANT (1) Xaa = modified
site when present, represents an amino acid, amino group, or
chemically modified amino terminus 491 Ala Gly Val Tyr Leu Leu Pro
Arg Arg Gly Glx 1 5 10 492 11 PRT Hepatitis C virus VARIANT (1) Xaa
= modified site when present, represents an amino acid, amino
group, or chemically modified amino terminus 492 Ala Val Tyr Leu
Leu Pro Arg Arg Gly Pro Glx 1 5 10 493 11 PRT Hepatitis C virus
VARIANT (1) Xaa = modified site when present, represents an amino
acid, amino group, or chemically modified amino terminus 493 Ala
Tyr Leu Leu Pro Arg Arg Gly Pro Arg Glx 1 5 10 494 11 PRT Hepatitis
C virus VARIANT (1) Xaa = modified site when present, represents an
amino acid, amino group, or chemically modified amino terminus 494
Ala Leu Leu Pro Arg Arg Gly Pro Arg Leu Glx 1 5 10 495 11 PRT
Hepatitis C virus VARIANT (1) Xaa = modified site when present,
represents an amino acid, amino group, or chemically modified amino
terminus 495 Ala Leu Pro Arg Arg Gly Pro Arg Leu Gly Glx 1 5 10 496
11 PRT Hepatitis C virus VARIANT (1) Xaa = modified site when
present, represents an amino acid, amino group, or chemically
modified amino terminus 496 Ala Pro Arg Arg Gly Pro Arg Leu Gly Val
Glx 1 5 10 497 11 PRT Hepatitis C virus VARIANT (1) Xaa = modified
site when present, represents an amino acid, amino group, or
chemically modified amino terminus 497 Ala Gly Pro Arg Leu Gly Val
Arg Ala Thr Glx 1 5 10 498 11 PRT Hepatitis C virus VARIANT (1) Xaa
= modified site when present, represents an amino acid, amino
group, or chemically modified amino terminus 498 Ala Pro Arg Leu
Gly Val Arg Ala Thr Arg Glx 1 5 10 499 11 PRT Hepatitis C virus
VARIANT (1) Xaa = modified site when present, represents an amino
acid, amino group, or chemically modified amino terminus 499 Ala
Arg Leu Gly Val Arg Ala Thr Arg Lys Glx 1 5 10 500 11 PRT Hepatitis
C virus VARIANT (1) Xaa = modified site when present, represents an
amino acid, amino group, or chemically modified amino terminus 500
Ala Glu Arg Ser Gln Pro Arg Gly Arg Arg Glx 1 5 10 501 11 PRT
Hepatitis C virus VARIANT (1) Xaa = modified site when present,
represents an amino acid, amino group, or chemically modified amino
terminus 501 Ala Arg Ser Gln Pro Arg Gly Arg Arg Gln Glx 1 5 10 502
11 PRT Hepatitis C virus VARIANT (1) Xaa = modified site when
present, represents an amino acid, amino group, or chemically
modified amino terminus 502 Ala Ser Gln Pro Arg Gly Arg Arg Gln Pro
Glx 1 5 10 503 11 PRT Hepatitis C virus VARIANT (1) Xaa = modified
site when present, represents an amino acid, amino group, or
chemically modified amino terminus 503 Ala Arg Gly Arg Arg Gln Pro
Ile Pro Lys Glx 1 5 10 504 11 PRT Hepatitis C virus VARIANT (1) Xaa
= modified site when present, represents an amino acid, amino
group, or chemically modified amino terminus 504 Ala Gly Arg Arg
Gln Pro Ile Pro Lys Val Glx 1 5 10 505 11 PRT Hepatitis C virus
VARIANT (1) Xaa = modified site when present, represents an amino
acid, amino group, or chemically modified amino terminus 505 Ala
Arg Arg Gln Pro Ile Pro Lys Val Arg Glx 1 5 10 506 11 PRT Hepatitis
C virus VARIANT (1) Xaa = modified site when present, represents an
amino acid, amino group, or chemically modified amino terminus 506
Ala Pro Ile Pro Lys Val Arg Arg Pro Glu Glx 1 5 10 507 11 PRT
Hepatitis C virus VARIANT (1) Xaa = modified site when present,
represents an amino acid, amino group, or chemically modified amino
terminus 507 Ala Pro Glu Gly Arg Thr Trp Ala Gln Pro Glx 1 5 10 508
11 PRT Hepatitis C virus VARIANT (1) Xaa = modified site when
present, represents an amino acid, amino group, or chemically
modified amino terminus 508 Ala Glu Gly Arg Thr Trp Ala Gln Pro Gly
Glx 1 5 10 509 11 PRT Hepatitis C virus VARIANT (1) Xaa = modified
site when present, represents an amino acid, amino group, or
chemically modified amino terminus 509 Ala Gly Arg Thr Trp Ala Gln
Pro Gly Tyr Glx 1 5 10 510 11 PRT Hepatitis C virus VARIANT (1) Xaa
= modified site when present, represents an amino acid, amino
group, or chemically modified amino terminus 510 Ala Arg Thr Trp
Ala Gln Pro Gly Tyr Pro Glx 1 5 10 511 11 PRT Hepatitis C virus
VARIANT (1) Xaa = modified site when present, represents an amino
acid, amino group, or chemically modified amino terminus 511 Ala
Thr Trp Ala Gln Pro Gly Tyr Pro Trp Glx 1 5 10 512 11 PRT Hepatitis
C virus VARIANT (1) Xaa = modified site when present, represents an
amino acid, amino group, or chemically modified amino terminus 512
Ala Trp Ala Gln Pro Gly Tyr Pro Trp Pro Glx 1 5 10 513 11 PRT
Hepatitis C virus VARIANT (1) Xaa = modified site when present,
represents an amino acid, amino group, or chemically modified amino
terminus 513 Ala Ala Gln Pro Gly Tyr Pro Trp Pro Leu Glx 1 5 10 514
11 PRT Hepatitis C virus VARIANT (1) Xaa = modified site when
present, represents an amino acid, amino group, or chemically
modified amino terminus 514 Ala Gln Pro Gly Tyr Pro Trp Pro Leu Tyr
Glx 1 5 10 515 11 PRT Hepatitis C virus VARIANT (1) Xaa = modified
site when present, represents an amino acid, amino group, or
chemically modified amino terminus 515 Ala Leu Ser Gly Lys Pro Ala
Ile Ile Pro Glx 1 5 10 516 11 PRT Hepatitis C virus VARIANT (1) Xaa
= modified site when present, represents an amino acid, amino
group, or chemically modified amino terminus 516 Ala Gly Lys Pro
Ala Ile Ile Pro Asp Arg Glx 1 5 10 517 11 PRT Hepatitis C virus
VARIANT (1) Xaa = modified site when present, represents an amino
acid, amino group, or chemically modified amino terminus 517 Ala
Pro Ala Ile Ile Pro Asp Arg Glu Val Glx 1 5 10 518 11 PRT Hepatitis
C virus VARIANT (1) Xaa = modified site when present, represents an
amino acid, amino group, or chemically modified amino terminus 518
Ala Ala Ile Ile Pro Asp Arg Glu Val Leu Glx 1 5 10 519 11 PRT
Hepatitis C virus VARIANT (1) Xaa = modified site when present,
represents an amino acid, amino group, or chemically modified amino
terminus 519 Ala Ile Ile Pro Asp Arg Glu Val Leu Tyr Glx
1 5 10 520 11 PRT Hepatitis C virus VARIANT (1) Xaa = modified site
when present, represents an amino acid, amino group, or chemically
modified amino terminus 520 Ala Ile Pro Asp Arg Glu Val Leu Tyr Arg
Glx 1 5 10 521 11 PRT Hepatitis C virus VARIANT (1) Xaa = modified
site when present, represents an amino acid, amino group, or
chemically modified amino terminus 521 Ala Pro Asp Arg Glu Val Leu
Tyr Arg Glu Glx 1 5 10 522 11 PRT Hepatitis C virus VARIANT (1) Xaa
= modified site when present, represents an amino acid, amino
group, or chemically modified amino terminus 522 Ala Asp Arg Glu
Val Leu Tyr Arg Glu Phe Glx 1 5 10 523 11 PRT Hepatitis C virus
VARIANT (1) Xaa = modified site when present, represents an amino
acid, amino group, or chemically modified amino terminus 523 Ala
Cys Ser Gln His Leu Pro Tyr Ile Glu Glx 1 5 10 524 11 PRT Hepatitis
C virus VARIANT (1) Xaa = modified site when present, represents an
amino acid, amino group, or chemically modified amino terminus 524
Ala Ser Gln His Leu Pro Tyr Ile Glu Gln Glx 1 5 10 525 11 PRT
Hepatitis C virus VARIANT (1) Xaa = modified site when present,
represents an amino acid, amino group, or chemically modified amino
terminus 525 Ala Gln His Leu Pro Tyr Ile Glu Gln Gly Glx 1 5 10 526
11 PRT Hepatitis C virus VARIANT (1) Xaa = modified site when
present, represents an amino acid, amino group, or chemically
modified amino terminus 526 Ala His Leu Pro Tyr Ile Glu Gln Gly Met
Glx 1 5 10 527 11 PRT Hepatitis C virus VARIANT (1) Xaa = modified
site when present, represents an amino acid, amino group, or
chemically modified amino terminus 527 Ala Leu Pro Tyr Ile Glu Gln
Gly Met Met Glx 1 5 10 528 11 PRT Hepatitis C virus VARIANT (1) Xaa
= modified site when present, represents an amino acid, amino
group, or chemically modified amino terminus 528 Ala Pro Tyr Ile
Glu Gln Gly Met Met Leu Glx 1 5 10 529 11 PRT Hepatitis C virus
VARIANT (1) Xaa = modified site when present, represents an amino
acid, amino group, or chemically modified amino terminus 529 Ala
Tyr Ile Glu Gln Gly Met Met Leu Ala Glx 1 5 10 530 11 PRT Hepatitis
C virus VARIANT (1) Xaa = modified site when present, represents an
amino acid, amino group, or chemically modified amino terminus 530
Ala Ile Glu Gln Gly Met Met Leu Ala Glu Glx 1 5 10 531 11 PRT
Hepatitis C virus VARIANT (1) Xaa = modified site when present,
represents an amino acid, amino group, or chemically modified amino
terminus 531 Ala Met Met Leu Ala Glu Gln Phe Lys Gln Glx 1 5 10 532
11 PRT Hepatitis C virus VARIANT (1) Xaa = modified site when
present, represents an amino acid, amino group, or chemically
modified amino terminus 532 Ala Met Leu Ala Glu Gln Phe Lys Gln Lys
Glx 1 5 10 533 11 PRT Hepatitis C virus VARIANT (1) Xaa = modified
site when present, represents an amino acid, amino group, or
chemically modified amino terminus 533 Ala Leu Ala Glu Gln Phe Lys
Gln Lys Ala Glx 1 5 10 534 11 PRT Hepatitis C virus VARIANT (1) Xaa
= modified site when present, represents an amino acid, amino
group, or chemically modified amino terminus 534 Ala Ala Glu Gln
Phe Lys Gln Lys Ala Leu Glx 1 5 10 535 11 PRT Hepatitis C virus
VARIANT (1) Xaa = modified site when present, represents an amino
acid, amino group, or chemically modified amino terminus 535 Ala
Glu Gln Phe Lys Gln Lys Ala Leu Gly Glx 1 5 10 536 11 PRT Hepatitis
C virus VARIANT (1) Xaa = modified site when present, represents an
amino acid, amino group, or chemically modified amino terminus 536
Ala Gln Phe Lys Gln Lys Ala Leu Gly Leu Glx 1 5 10 537 11 PRT
Hepatitis C virus VARIANT (1) Xaa = modified site when present,
represents an amino acid, amino group, or chemically modified amino
terminus 537 Ala Phe Lys Gln Lys Ala Leu Gly Leu Leu Glx 1 5 10 538
11 PRT Hepatitis C virus VARIANT (1) Xaa = modified site when
present, represents an amino acid, amino group, or chemically
modified amino terminus 538 Ala Lys Gln Lys Ala Leu Gly Leu Leu Gln
Glx 1 5 10 539 11 PRT Hepatitis C virus VARIANT (1) Xaa = modified
site when present, represents an amino acid, amino group, or
chemically modified amino terminus 539 Ala Gln Lys Ala Leu Gly Leu
Leu Gln Thr Glx 1 5 10 540 11 PRT Hepatitis C virus VARIANT (1) Xaa
= modified site when present, represents an amino acid, amino
group, or chemically modified amino terminus 540 Ala Lys Ala Leu
Gly Leu Leu Gln Thr Ala Glx 1 5 10 541 11 PRT Hepatitis C virus
VARIANT (1) Xaa = modified site when present, represents an amino
acid, amino group, or chemically modified amino terminus 541 Ala
Ala Leu Gly Leu Leu Gln Thr Ala Ser Glx 1 5 10 542 11 PRT Hepatitis
C virus VARIANT (1) Xaa = modified site when present, represents an
amino acid, amino group, or chemically modified amino terminus 542
Ala Leu Gly Leu Leu Gln Thr Ala Ser Arg Glx 1 5 10 543 11 PRT
Hepatitis C virus VARIANT (1) Xaa = modified site when present,
represents an amino acid, amino group, or chemically modified amino
terminus 543 Ala Gly Leu Leu Gln Thr Ala Ser Arg Gln Glx 1 5 10 544
11 PRT Hepatitis C virus VARIANT (1) Xaa = modified site when
present, represents an amino acid, amino group, or chemically
modified amino terminus 544 Ala Leu Leu Gln Thr Ala Ser Arg Gln Ala
Glx 1 5 10 545 11 PRT Hepatitis C virus VARIANT (1) Xaa = modified
site when present, represents an amino acid, amino group, or
chemically modified amino terminus 545 Ala Ser Val Pro Ala Glu Ile
Leu Arg Lys Glx 1 5 10 546 11 PRT Hepatitis C virus VARIANT (1) Xaa
= modified site when present, represents an amino acid, amino
group, or chemically modified amino terminus 546 Ala Val Pro Ala
Glu Ile Leu Arg Lys Ser Glx 1 5 10 547 11 PRT Hepatitis C virus
VARIANT (1) Xaa = modified site when present, represents an amino
acid, amino group, or chemically modified amino terminus 547 Ala
Pro Ala Glu Ile Leu Arg Lys Ser Arg Glx 1 5 10 548 11 PRT Hepatitis
C virus VARIANT (1) Xaa = modified site when present, represents an
amino acid, amino group, or chemically modified amino terminus 548
Ala Ala Glu Ile Leu Arg Lys Ser Arg Arg Glx 1 5 10 549 11 PRT
Hepatitis C virus VARIANT (1) Xaa = modified site when present,
represents an amino acid, amino group, or chemically modified amino
terminus 549 Ala Glu Ile Leu Arg Lys Ser Arg Arg Phe Glx 1 5 10 550
11 PRT Hepatitis C virus VARIANT (1) Xaa = modified site when
present, represents an amino acid, amino group, or chemically
modified amino terminus 550 Ala Phe Ala Gln Ala Leu Pro Val Trp Ala
Glx 1 5 10 551 11 PRT Hepatitis C virus VARIANT (1) Xaa = modified
site when present, represents an amino acid, amino group, or
chemically modified amino terminus 551 Ala Ala Gln Ala Leu Pro Val
Trp Ala Arg Glx 1 5 10 552 11 PRT Hepatitis C virus VARIANT (1) Xaa
= modified site when present, represents an amino acid, amino
group, or chemically modified amino terminus 552 Ala Gln Ala Leu
Pro Val Trp Ala Arg Pro Glx 1 5 10 553 11 PRT Hepatitis C virus
VARIANT (1) Xaa = modified site when present, represents an amino
acid, amino group, or chemically modified amino terminus 553 Ala
Ala Leu Pro Val Trp Ala Arg Pro Asp Glx 1 5 10 554 11 PRT Hepatitis
C virus VARIANT (1) Xaa = modified site when present, represents an
amino acid, amino group, or chemically modified amino terminus 554
Ala Val Trp Ala Arg Pro Asp Tyr Asn Pro Glx 1 5 10 555 11 PRT
Hepatitis C virus VARIANT (1) Xaa = modified site when present,
represents an amino acid, amino group, or chemically modified amino
terminus 555 Ala Trp Ala Arg Pro Asp Tyr Asn Pro Pro Glx 1 5 10 556
11 PRT Hepatitis C virus VARIANT (1) Xaa = modified site when
present, represents an amino acid, amino group, or chemically
modified amino terminus 556 Ala Ala Arg Pro Asp Tyr Asn Pro Pro Leu
Glx 1 5 10 557 11 PRT Hepatitis C virus VARIANT (1) Xaa = modified
site when present, represents an amino acid, amino group, or
chemically modified amino terminus 557 Ala Arg Pro Asp Tyr Asn Pro
Pro Leu Val Glx 1 5 10 558 11 PRT Hepatitis C virus VARIANT (1) Xaa
= modified site when present, represents an amino acid, amino
group, or chemically modified amino terminus 558 Ala Pro Asp Tyr
Asn Pro Pro Leu Val Glu Glx 1 5 10 559 11 PRT Hepatitis C virus
VARIANT (1) Xaa = modified site when present, represents an amino
acid, amino group, or chemically modified amino terminus 559 Ala
Pro Pro Leu Val Glu Thr Trp Lys Lys Glx 1 5 10 560 11 PRT Hepatitis
C virus VARIANT (1) Xaa = modified site when present, represents an
amino acid, amino group, or chemically modified amino terminus 560
Ala Pro Leu Val Glu Thr Trp Lys Lys Pro Glx 1 5 10 561 11 PRT
Hepatitis C virus VARIANT (1) Xaa = modified site when present,
represents an amino acid, amino group, or chemically modified amino
terminus 561 Ala Leu Val Glu Thr Trp Lys Lys Pro Asp Glx 1 5 10 562
11 PRT Hepatitis C virus VARIANT (1) Xaa = modified site when
present, represents an amino acid, amino group, or chemically
modified amino terminus 562 Ala Trp Glu Thr Trp Lys Lys Pro Asp Tyr
Glx 1 5 10 563 11 PRT Hepatitis C virus VARIANT (1) Xaa = modified
site when present, represents an amino acid, amino group, or
chemically modified amino terminus 563 Ala Glu Thr Trp Lys Lys Pro
Asp Tyr Glu Glx 1 5 10 564 11 PRT Hepatitis C virus VARIANT (1) Xaa
= modified site when present, represents an amino acid, amino
group, or chemically modified amino terminus 564 Ala Thr Trp Lys
Lys Pro Asp Tyr Glu Pro Glx 1 5 10 565 11 PRT Hepatitis C virus
VARIANT (1) Xaa = modified site when present, represents an amino
acid, amino group, or chemically modified amino terminus 565 Ala
Trp Lys Lys Pro Asp Tyr Glu Pro Pro Glx 1 5 10 566 11 PRT Hepatitis
C virus VARIANT (1) Xaa = modified site when present, represents an
amino acid, amino group, or chemically modified amino terminus 566
Ala Lys Lys Pro Asp Tyr Glu Pro Pro Val Glx 1 5 10 567 11 PRT
Hepatitis C virus VARIANT (1) Xaa = modified site when present,
represents an amino acid, amino group, or chemically modified amino
terminus 567 Ala Lys Pro Asp Tyr Glu Pro Pro Val Val Glx 1 5 10 568
11 PRT Hepatitis C virus VARIANT (1) Xaa = modified site when
present, represents an amino acid, amino group, or chemically
modified amino terminus 568 Ala Pro Asp Tyr Glu Pro Pro Val Val His
Glx 1 5 10 569 11 PRT Hepatitis C virus VARIANT (1) Xaa = modified
site when present, represents an amino acid, amino group, or
chemically modified amino terminus 569 Ala Asp Tyr Glu Pro Pro Val
Val His Gly Glx 1 5 10 570 11 PRT Hepatitis C virus VARIANT (1) Xaa
= modified site when present, represents an amino acid, amino
group, or chemically modified amino terminus 570 Ala Tyr Glu Pro
Pro Val Val His Gly Cys Glx 1 5 10 571 11 PRT Hepatitis C virus
VARIANT (1) Xaa = modified site when present, represents an amino
acid, amino group, or chemically modified amino terminus 571 Ala
Pro Pro Val Val His Gly Cys Pro Leu Glx 1 5 10 572 11 PRT Hepatitis
C virus VARIANT (1) Xaa = modified site when present, represents an
amino acid, amino group, or chemically modified amino terminus 572
Ala Pro Val Val His Gly Cys Pro Leu Pro Glx 1 5 10 573 11 PRT
Hepatitis C virus VARIANT (1) Xaa = modified site when present,
represents an amino acid, amino group, or chemically modified amino
terminus 573 Ala Val Val His Gly Cys Pro Leu Pro Pro Glx 1 5 10 574
11 PRT Hepatitis C virus VARIANT (1) Xaa = modified site when
present, represents an amino acid, amino group, or chemically
modified amino terminus 574 Ala Val His Gly Cys Pro Leu Pro Pro Lys
Glx 1 5 10 575 11 PRT Hepatitis C virus VARIANT (1) Xaa = modified
site when present, represents an amino acid, amino group, or
chemically modified amino terminus 575 Ala His Gly Cys Pro Leu Pro
Pro Lys Ser Glx 1 5 10 576 11 PRT Hepatitis C virus VARIANT (1) Xaa
= modified site when present, represents an amino acid, amino
group, or chemically modified amino terminus 576 Ala Ser Pro Pro
Val Pro Pro Pro Arg Lys Glx 1 5 10 577 8 PRT Hepatitis C virus
VARIANT (1) Xaa = modified site when present, represents an amino
acid, amino group, or chemically modified amino terminus 577 Ala
Pro Gln Arg Lys Thr Lys Glx 1 5 578 8 PRT Hepatitis C virus VARIANT
(1) Xaa = modified site when present, represents an amino acid,
amino group, or chemically modified amino terminus 578 Ala Pro Gln
Arg Lys Thr Lys Glx 1 5 579 9 PRT Hepatitis C virus VARIANT (1) Xaa
= modified site when present, represents an amino acid, amino
group, or chemically modified amino terminus 579 Ala Pro Gln Asp
Val Lys Phe Pro Glx 1 5 580 8 PRT Hepatitis C virus VARIANT (1) Xaa
= modified site when present, represents an amino acid, amino
group, or chemically modified amino terminus 580 Ala Tyr Leu Leu
Pro Arg Arg Glx 1 5 581 9 PRT Hepatitis C virus VARIANT (1) Xaa =
modified site when present, represents an amino acid, amino group,
or chemically modified amino terminus 581 Ala Pro Arg Arg Gly Pro
Arg Leu Glx 1 5 582 9 PRT Hepatitis C virus VARIANT (1) Xaa =
modified site when present, represents an amino acid, amino group,
or chemically modified amino terminus 582 Ala Arg Leu Gly Val Arg
Ala Thr Glx 1 5 583 9 PRT Hepatitis C virus VARIANT (1) Xaa =
modified site when present, represents an amino acid, amino group,
or chemically modified amino terminus 583 Ala Ser Gln Pro Arg Gly
Arg Arg Glx 1 5 584 9 PRT Hepatitis C virus VARIANT (1) Xaa =
modified site when present, represents an amino acid, amino group,
or chemically modified amino terminus 584 Ala Arg Arg Gln Pro Ile
Pro Lys Glx 1 5 585 8 PRT Hepatitis C virus VARIANT (1) Xaa =
modified site when present, represents an amino acid, amino group,
or chemically modified amino terminus 585 Ala Arg Thr Trp Ala Gln
Pro Glx 1 5 586 10 PRT Hepatitis C virus VARIANT (1) Xaa = modified
site when present, represents an amino acid, amino group, or
chemically modified amino terminus 586 Ala Gln Pro Gly Tyr Pro Trp
Pro Leu Glx 1 5 10 587 8 PRT Hepatitis C virus VARIANT (1) Xaa =
modified site when present, represents an amino acid, amino group,
or chemically modified amino terminus 587 Ala Pro Asp Arg Glu Val
Leu Glx 1 5 588 8 PRT Hepatitis C virus VARIANT (1) Xaa = modified
site when present, represents an amino acid, amino group, or
chemically modified amino terminus 588 Ala His Leu Pro Tyr Ile Glu
Glx 1 5 589 10 PRT Hepatitis C virus VARIANT (1) Xaa = modified
site when present, represents an amino acid, amino group, or
chemically modified amino terminus 589 Ala Tyr Ile Glu Gln Gly Met
Met Leu Glx 1 5 10 590 9 PRT Hepatitis C virus VARIANT (1) Xaa =
modified site when present, represents an amino acid, amino group,
or chemically modified amino terminus 590 Ala Ala Glu Gln Phe Lys
Gln Lys Glx 1 5 591 8 PRT Hepatitis C virus VARIANT (1) Xaa =
modified site when present, represents an amino acid, amino group,
or chemically modified amino terminus 591 Ala Lys Gln Lys Ala Leu
Gly Glx 1 5 592 9 PRT Hepatitis C virus VARIANT (1) Xaa = modified
site when present, represents an amino acid, amino group, or
chemically modified amino terminus 592 Ala Leu Gly Leu Leu Gln Thr
Ala Glx 1 5 593 9 PRT Hepatitis C virus VARIANT (1) Xaa = modified
site when present, represents an amino acid, amino group, or
chemically modified amino
terminus 593 Ala Pro Ala Glu Ile Leu Arg Lys Glx 1 5 594 9 PRT
Hepatitis C virus VARIANT (1) Xaa = modified site when present,
represents an amino acid, amino group, or chemically modified amino
terminus 594 Ala Glu Ile Leu Arg Lys Ser Arg Glx 1 5 595 9 PRT
Hepatitis C virus VARIANT (1) Xaa = modified site when present,
represents an amino acid, amino group, or chemically modified amino
terminus 595 Ala Gln Ala Leu Pro Val Trp Ala Glx 1 5 596 8 PRT
Hepatitis C virus VARIANT (1) Xaa = modified site when present,
represents an amino acid, amino group, or chemically modified amino
terminus 596 Ala Pro Asp Tyr Asn Pro Pro Glx 1 5 597 9 PRT
Hepatitis C virus VARIANT (1) Xaa = modified site when present,
represents an amino acid, amino group, or chemically modified amino
terminus 597 Ala Leu Val Glu Thr Trp Lys Lys Glx 1 5 598 8 PRT
Hepatitis C virus VARIANT (1) Xaa = modified site when present,
represents an amino acid, amino group, or chemically modified amino
terminus 598 Ala Asp Tyr Glu Pro Pro Val Glx 1 5 599 7 PRT
Hepatitis C virus VARIANT (1) Xaa = modified site when present,
represents an amino acid, amino group, or chemically modified amino
terminus 599 Ala His Gly Cys Pro Leu Glx 1 5 600 20 PRT Hepatitis C
virus 600 Gly Arg Thr Trp Ala Gln Pro Gly Tyr Pro Trp Pro Leu Tyr
Gly Asn 1 5 10 15 Glu Gly Cys Gly 20
* * * * *