U.S. patent application number 15/093615 was filed with the patent office on 2017-03-02 for immunogen platform.
The applicant listed for this patent is ChronTech Pharma AB. Invention is credited to Lars Frelin, Matti Sallberg, Jonas Soderholm.
Application Number | 20170058003 15/093615 |
Document ID | / |
Family ID | 40351231 |
Filed Date | 2017-03-02 |
United States Patent
Application |
20170058003 |
Kind Code |
A1 |
Sallberg; Matti ; et
al. |
March 2, 2017 |
IMMUNOGEN PLATFORM
Abstract
Aspects of the present invention relate to chimeric polypeptides
including HCV NS3/4A sequences and T-cell epitopes. Embodiments
include nucleic acids encoding the chimeric NS3/4A polypeptides,
the encoded polypeptides, compositions containing said nucleic
acids, compositions containing said chimeric polypeptides, as well
as methods of making and using the aforementioned compositions
including, but not limited to medicaments and vaccines.
Inventors: |
Sallberg; Matti; (Stockholm,
SE) ; Soderholm; Jonas; (Linghem, SE) ;
Frelin; Lars; (Alvsjo, SE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ChronTech Pharma AB |
Huddinge |
|
SE |
|
|
Family ID: |
40351231 |
Appl. No.: |
15/093615 |
Filed: |
April 7, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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14527669 |
Oct 29, 2014 |
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15093615 |
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13178373 |
Jul 7, 2011 |
8883169 |
|
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14527669 |
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12192776 |
Aug 15, 2008 |
8071561 |
|
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13178373 |
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61047076 |
Apr 22, 2008 |
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60956326 |
Aug 16, 2007 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2039/55566
20130101; C07K 2319/00 20130101; A61K 2039/57 20130101; C07K 14/005
20130101; A61K 2039/575 20130101; C12N 2730/10134 20130101; A61K
39/00 20130101; C12N 2770/24222 20130101; A61K 39/12 20130101; C07K
2319/50 20130101; A61K 39/292 20130101; A61K 2039/572 20130101;
C12N 7/00 20130101; A61K 2039/55511 20130101; A61K 39/29 20130101;
C12N 2770/24234 20130101; A61K 39/39 20130101; C12N 2730/10122
20130101; A61K 2039/53 20130101 |
International
Class: |
C07K 14/005 20060101
C07K014/005; A61K 39/29 20060101 A61K039/29; A61K 39/39 20060101
A61K039/39; C12N 7/00 20060101 C12N007/00 |
Claims
1. (canceled)
2. An immunogenic composition comprising a nucleic acid molecule
that comprises a nucleotide sequence encoding a heterologous
chimeric hepatitis viral antigen comprising hepatitis C virus (HCV)
NS3/4A antigen fused to a hepatitis B virus (HBV) core antigen,
wherein the nucleotide sequence is modified to encode an amino acid
sequence that comprises one or more protease cleavage site
insertions at one or more non-naturally occurring positions and
said immunogenic compositions induces an effective immune response
specific for one or more epitopes of said heterologous viral
antigen.
3. The immunogenic composition of claim 2, wherein the immune
response is a humoral antibody response.
4. A method of inducing an immune response to an HCV antigen, the
method comprising: identifying a patient in need of an induced
immune response to HCV antigen; and administering an amount of
composition effective to induce an immune response to said patient,
wherein the composition comprises a nucleic acid molecule that
comprises a nucleotide sequence encoding a heterologous chimeric
hepatitis viral antigen comprising hepatitis C virus (HCV) NS3/4A
antigen fused to a hepatitis B virus (HBV) core antigen, wherein
the nucleotide sequence is modified to encode an amino acid
sequence that comprises one or more protease cleavage site
insertions at one or more non-naturally occurring positions and
said immunogenic compositions induces an effective immune response
specific for one or more epitopes of said heterologous viral
antigen.
5. The method of claim 4, further comprising co-administering an
adjuvant with said composition, wherein the adjuvant is
ribavirin.
6. The method of claim 4, wherein the induced immune response is a
humoral antibody response.
Description
REFERENCE TO SEQUENCE LISTING
[0001] The present application is being filed along with a Sequence
Listing in electronic format. The Sequence Listing is provided as a
file entitled SeqList-TRIPEP_111C3.txt, created Apr. 7, 2016, which
is 4893 KB in size. The information in the electronic format of the
Sequence Listing is incorporated herein by reference in its
entirety.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0002] This application is a continuation of and claims the benefit
of priority to U.S. patent application Ser. No. 14/527,669, filed
Oct. 29, 2014, which is a continuation of and claims priority to
U.S. patent application Ser. No. 13/178,373, now U.S. Pat. No.
8,883,169, filed Jul. 7, 2011, which is a continuation of and
claims priority to U.S. patent application Ser. No. 12/192,776, now
U.S. Pat. No. 8,071,561, filed Aug. 15, 2008, which claims priority
to U.S. Provisional Application No. 60/956,326, filed Aug. 16, 2007
and also claims priority to U.S. Provisional Application No.
61/047,076, filed Apr. 22, 2008. All of the aforementioned
applications are hereby expressly incorporated by reference in
their entireties.
BACKGROUND
[0003] Traditionally, vaccines have been based on live attenuated
or inactivated pathogens. These strategies are inefficient,
however, largely because of the antigenic variability of pathogens
(e.g., viruses). Several peptide vaccines that comprise antigenic
peptides or peptide fragments of pathogens have been developed.
Conserved peptide fragments are less likely to exhibit antigenic
variability and can overcome some of the problems associated with
traditional peptides. Accordingly, subunit vaccines have been
developed, which target conserved regions of pathogens. Synthetic
peptide vaccines tend to be poorly immunogenic, however. The poor
immunogenicity of synthetic peptide vaccines may be attributed to
the fact that although these types of vaccines induce humoral
antibody responses, they are less likely to induce cell-mediated
responses.
[0004] Several investigators have sought to improve the
antigenicity of synthetic peptide vaccines. For example, Klein et
al. describe the engineering of chimeric proteins that comprise an
immunogenic region of a protein from a first antigen linked to an
immunogenic region from a second pathogen. (See, U.S. Pat. Nos.
6,033,668; 6,017,539; 5,998,169; and 5,968,776). Others have sought
to create chimeric proteins that couple B-cell epitopes to
universal T-cell epitopes in order to improve the immune response.
(See, e.g., U.S. Pat. No. 5,114,713). Russell-Jones et al. (U.S.
Pat. No. 5,928,644) also disclose T-cell epitopes derived from the
TraT protein of Escherichia coli, which are used to produce hybrid
molecules so as to generate an immune response to parasites,
soluble factors (e.g., LSH) and viruses. Further, Ruslan (U.S.
Patent Application Publication No. 20030232055) discloses the
manufacture of vaccines based on PAMPs and immunogenic antigens.
Despite these advances, the development of compositions and methods
that improve the antigenicity of immunogens is manifest.
SUMMARY OF THE INVENTION
[0005] Several embodiments described herein concern compositions
and methods that are useful for the generation, enhancement, or
improvement of an immune response to a target antigen. Many
platforms for the presentation of antigens are provided. These
platforms are particularly useful for nucleic acid-based immunogens
(e.g., DNA vaccines). It has been discovered that the hepatitis C
virus (HCV) nonstructural protein 3 (NS3) and nonstructural protein
4A (NS4A), collectively NS3/4A, and fragments of this fusion
protein (e.g., fragments that retain the protease domain, protease
cleavage site, and/or the helicase domain) or a nucleic acid
encoding these proteins are useful platforms to present antigens
(e.g., nucleic acids encoding a T cell epitope, such as a CTL or
HTL domain) so as to generate a potent immune response to the
associated antigen.
[0006] One aim of using NS3/4A as a carrier or adjuvant is to
effectively provide T helper cells access to a fused antigen,
thereby enhancing the immune response to the fused antigen. In
addition, in some embodiments it is desired to have an active, or
highly active, NS3/4A protease since an enhanced or altered
protease activity can have adjuvant effects that improve the immune
response to the fused gene. Moreover, the active NS3/4A protease
can be used to cleave the fused protein (e.g., a heterologous
antigen), especially when it contains inserted heterologous
protease cleavage sites, into smaller fragments to enhance
processing and to ensure that the fused protein will not resemble
its native structure. For certain conditions or diseases it can be
desirable to use the fused protein in a way that is structurally
different from the native form since the native form of the protein
may have properties that are at an immunogenic disadvantage. It is
envisioned that the introduction of foreign protease cleavage sites
in the fusion protein (e.g., a peptide antigen) induce protein
cleavage into small fragments that can enhance processing.
Additionally, if the natural sequence has been changed the cleavage
at the introduced sites can ensure that no new, artificial
junctional T cell epitopes are generated.
[0007] Accordingly, embodiments disclosed herein include
compositions that comprise an isolated nucleic acid that encodes a
chimeric Hepatitis C virus (HCV) NS3/4A polypeptide or a fragment
thereof, which comprises a sequence that encodes an antigen
(preferably a non-HCV epitope or, when an HCV epitope is used, the
antigen is not in a position on the NS3/4A peptide or NS3/4A
nucleic acid or fragment thereof that is naturally occurring). The
immunogenic sequence (e.g., a nucleic acid encoding an antigen) can
be inserted within the NS3/4A nucleic acid or NS3/4A peptide or
attached thereto (e.g., the antigen-encoding nucleic acid can be
inserted within the nucleic acid encoding NS3 and/or NS4A domain at
a location outside of or within the protease domain, helicase
domain, or protease cleavage site or said sequence encoding said
antigen can be flanking the 5' or 3' end of the nucleic acid
encoding said HCV NS3/4A peptide, such as juxtaposed to the 5' or
3' end).
[0008] Embodiments also include an antigenic or immunogenic peptide
(e.g., a peptide of a pathogen, bacteria, toxin, virus, or cancer
cell antigen) joined to (e.g., flanking or juxtaposed to) or
affixed within NS3/4A peptide or a fragment thereof, which peptide
fragment can comprise at least, equal to, greater than, less than,
or any number in between 3, 5, 10, 20, 50, 100, 150, 200, 250, 300,
350, 400, 500, 700, 1000, 1200, or 1500 consecutive amino acids of
a natural or synthetic NS3/4A polypeptide (e.g., a naturally
occurring isotype of NS3/4A or a codon-optimized or otherwise
modified NS3/4A polypeptide (e.g., consensus NS3/4A sequences
generated from 2 or more isotypes), such as SEQ. ID. Nos. 2 and
36).
[0009] More embodiments include a nucleic acid that encodes an
antigenic or immunogenic peptide (e.g., a nucleic acid encoding a
peptide of a pathogen, bacteria, toxin, virus, or cancer cell
antigen) joined to or affixed within a nucleic acid encoding an
NS3/4A peptide or a fragment thereof (e.g., the nucleic acid that
encodes said antigenic or immunogenic peptide can be inserted
within or joined to (e.g., flanking or juxtaposed to) an isolated
but naturally occurring NS3/4A nucleic acid or a synthetic NS3/4A
nucleic acid (e.g., a codon-optimized NS3/4A nucleic acid) or a
fragment of these nucleic acids (e.g., the NS3/4a nucleic acid
fragment can comprise, consist of, or consist essentially of about
at least, equal to, greater than, less than, or any number in
between 9, 15, 30, 50, 75, 100, 125, 150, 175, 200, 250, 300, 400,
500, 600, 700, 800, 900, 1000, 1200, 1500, 2000, or 2500
consecutive nucleotides of a nucleic acid sequence that encodes an
isolated but natural NS3/4A polypeptide or synthetic NS3/4A
polypeptide (e.g., SEQ. ID. Nos. 1 and 35). Polypeptides encoded by
said nucleic acids are also embodiments.
[0010] In some embodiments, the antigenic or immunogenic peptide is
a T cell epitope (TCE), such as a CTL epitope or an HTL epitope, or
the antigenic or immunogenic nucleic acid encodes a TCE, such as a
CTL epitope or HTL epitope. As above, the TCE can be inserted
within or flanking (e.g., juxtaposed to) the NS3/4A peptide or
nucleic acids encoding the NS3/4A peptide or fragments of these
peptides and nucleic acids, as described above, such that said
chimeric sequences are or encode chimeric NS3/4A polypeptides or
fragments thereof with TCEs inserted within or flanking (e.g.,
juxtaposed to) the NS3/4A polypeptide sequences. Preferably, the
nucleic acid encoding the TCE, and the encoded TCE, is located at a
position that is not naturally occurring in HCV, when said TCE is
an HCV epitope. Desirably, the encoded NS3/4A chimeric polypeptide
retains catalytic activity (e.g., protease and/or helicase
activity).
[0011] Accordingly, several embodiments include a nucleic acid that
encodes a TCE (e.g., a CTL or HTL of a pathogen, bacteria, virus,
toxin, or of a cancer cell) inserted within or flanking (e.g.,
juxtaposed to) a nucleic acid encoding an NS3/4A polypeptide (e.g.,
SEQ. ID. Nos. 1 or 35) or a nucleic acid encoding a fragment of an
NS3/4A polypeptide (e.g., a fragment of SEQ. ID. Nos. 1 or 35),
preferably a fragment that retains protease and/or helicase
activity, wherein said fragment can comprise, consist of, or
consist essentially of about at least, equal to, greater than, less
than, or any number in between 9, 15, 30, 50, 75, 100, 125, 150,
175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1500,
2000, or 2500 consecutive nucleotides of a nucleic acid sequence
that encodes an isolated but natural NS3/4A polypeptide or
synthetic NS3/4A polypeptide (e.g., SEQ. ID. Nos. 1 or 35), such
that when said TCE is a sequence that encodes an HCV TCE, said HCV
TCE is in a position on the nucleic acid or polypeptide encoded by
said nucleic acid that is not naturally occurring. Polypeptides
encoding said nucleic acid embodiments are also aspects of the
invention.
[0012] Optionally, the isolated nucleic acids can also encode a
linker sequence and/or a sequence that promotes adjuvant activity
(e.g., a stimulatory TCE, a plurality of immune stimulatory di
nucleotides, such as CpG, or an RNA binding domain). For example,
in some embodiments, the linker sequence or adjuvant sequence
flanks (e.g., juxtaposed to) at least one end of the encoded TCE or
antigen. Preferably, the nucleic acid encodes a linker comprising
one to six alanine and/or glycine residues flanking or juxtaposed
to at least one of a TCE, for example, between TCE sequences and
N33/4A sequences. Polypeptides encoded by any of the nucleic acids
provided herein are also embodiments.
[0013] In some embodiments, nucleic acids encoding an antigen, TCE,
antigen and linker, TCE and linker, antigen and adjuvant sequence,
TCE and adjuvant sequence, antigen and linker and adjuvant
sequence, or TCE and linker and adjuvant sequence are inserted
within a nucleic acid encoding an HCV NS3/4A polypeptide (e.g., SEQ
ID NOs: 1 or 35) or fragment thereof (e.g., the NS3/4a nucleic acid
fragment can comprise, consist of, or consist essentially of about
at least, equal to, greater than, less than, or any number in
between 9, 15, 30, 50, 75, 100, 125, 150, 175, 200, 250, 300, 400,
500, 600, 700, 800, 900, 1000, 1200, 1500, or 2000 consecutive
nucleotides of an NS3 and/or NS4A sequence, such as SEQ ID NOs: 1
or 35). Such embodiments can be used as DNA immunogens, which can
be delivered to a subject in need of an immune response to the
antigen contained therein transdermally (e.g., a transdermal oil or
patch), by injection (e.g., hypodermic or ballistic),
electroporation (e.g., gene gun, microneedle, needless, exvivo,
MedPulsar.TM., electroporation with needles or following needleless
injection), or any combination thereof. Alternatively, polypeptides
encoded by said DNA immunogens can be delivered to a subject in
need of an immune response to the antigen. For example, in some
embodiments, a nucleic acid encoding an antigen, TCE, antigen and
linker, TCE and linker, antigen and adjuvant sequence, TCE and
adjuvant sequence, antigen and linker and adjuvant sequence, or TCE
and linker and adjuvant sequence is inserted between the codons of
an NS3/4A nucleic acid at sites that do not interrupt protease
activity and/or helicase activity such that the expressed fusion
peptide retains such catalytic activity and the nucleic acid
immunogen is delivered to a subject in need of an immune response
to said antigen or TCE by an electroporation system that comprises
a needle injection system (e.g., Medpulser.TM.). Again, aspects
disclosed herein include polypeptides that are encoded by said
nucleic acids.
[0014] That is, in some embodiments, antigen, TCE, antigen and
linker, TCE and linker, antigen and adjuvant sequence, TCE and
adjuvant sequence, antigen and linker and adjuvant sequence, or TCE
and linker and adjuvant sequence are inserted between nucleotides 3
and 4, 6 and 7, 9 and 10, 12 and 13, 15 and 16, 18 and 19, 21 and
22, 24 and 25, 27 and 28, 30 and 31, 33 and 34, 36 and 37, 39 and
40, 42 and 43, 45 and 46, 48 and 49, 51 and 52, 54 and 55, 57 and
58, 60 and 61, 63 and 64, 66 and 67, 69 and 70, 72 and 73, 75 and
76, 78 and 79, 81 and 82, 84 and 85, 87 and 88, 90 and 91, 93 and
94, 96 and 97, 99 and 100, 102 and 103, 105 and 106, 108 and 109,
111 and 112, 114 and 115, 117 and 118, 120 and 121, 123 and 124,
126 and 127, 129 and 130, 132 and 133, 125 and 136, 138 and 139,
141 and 142, 144 and 145, 147 and 148, 150 and 151, 153 and 154,
156 and 157, 159 and 160, 162 and 163, 165 and 166, 168 and 169,
171 and 172, 174 and 175, 177 and 178, 180 and 181, 183 and 184,
186 and 187, 189 and 19, 192 and 193, 195 and 196, 198 and 199, 201
and 202, 204 and 205, 207 and 208, 210 and 211, 213 and 214, 216
and 217, 219 and 220, 222 and 223, 225 and 226, 228 and 229, 231
and 232, 234 and 235, 237 and 238, 240 and 241, 243 and 244, 246
and 247, 249 and 250, 252 and 253, 255 and 256, 258 and 259, 261
and 262, 264 and 265, 267 and 268, 270 and 271, 273 and 274, 276
and 277, 279 and 280, 282 and 283, 285 and 286, 288 and 289, 291
and 292, 294 and 295, 297 and 298, 300 and 301, 303 and 304, 306
and 307, 309 and 310, 312 and 313, 315 and 316, 318 and 319, 321
and 322, 324 and 325, 327 and 328, 330 and 331, 333 and 334, 336
and 337, 339 and 340, 342 and 343, 345 and 346, 348 and 349, 351
and 352, 354 and 355, 357 and 358, 360 and 361, 363 and 364, 366
and 367, 369 and 370, 372 and 373, 375 and 376, 378 and 379, 381
and 382, 24 and 385, 387 and 388, 390 and 391, 393 and 394, 396 and
397, 399 and 400, 402 and 403, 405 and 406, 408 and 409, 411 and
412, 414 and 415, 417 and 418, 420 and 421, 423 and 424, 426 and
427, 429 and 430, 432 and 433, 435 and 436, 438 and 439, 441 and
442, 444 and 445, 447 and 448, 450 and 451, 453 and 454, 456 and
457, 459 and 460, 462 and 463, 465 and 466, 468 and 469, 471 and
472, 474 and 475, 477 and 478, 480 and 481, 483 and 484, 486 and
487, 489 and 490, 492 and 493, 495 and 496, 498 and 499, 501 and
502, 504 and 505, 507 and 508, 510 and 511, 513 and 514, 516 and
517, 519 and 520, 522 and 523, 525 and 526, 528 and 529, 531 and
532, 534 and 535, 537 and 538, 540 and 541, 543 and 544, 546 and
547, 549 and 550, 552 and 553, 555 and 556, 558 and 559, 561 and
562, 564 and 565, 567 and 568, 570 and 571, 573 and 574, 576 and
577, 579 and 580, 582 and 583, 585 and 586, 588 and 589, 591 and
592, 594 and 595, 597 and 598, 600 and 601, 603 and 604, 606 and
607, 609 and 610, 612 and 613, 615 and 616, 618 and 619, 621 and
622, 624 and 625, 627 and 628, 630 and 631, 633 and 634, 636 and
637, 639 and 640, 642 and 643, 645 and 646, 648 and 649, 651 and
652, 654 and 655, 657 and 658, 660 and 661, 663 and 664, 666 and
667, 669 and 670, 672 and 673, 675 and 676, 678 and 679, 681 and
682, 684 and 685, 687 and 688, 690 and 691, 693 and 694, 696 and
697, 699 and 700, 702 and 703, 705 and 706, 708 and 709, 711 and
712, 714 and 715, 717 and 718, 720 and 721, 723 and 724, 726 and
727, 729 and 730, 732 and 733, 735 and 736, 738 and 739, 741 and
742, 744 and 745, 747 and 748, 750 and 751, 753 and 754, 756 and
757, 759 and 760, 762 and 763, 765 and 766, 768 and 769, 771 and
772, 774 and 775, 777 and 778, 780 and 781, 783 and 784, 786 and
787, 789 and 790, 792 and 793, 795 and 796, 798 and 799, 801 and
802, 804 and 805, 807 and 808, 810 and 811, 813 and 814, 816 and
817, 819 and 820, 822 and 823, 825 and 826, 828 and 829, 831 and
832, 834 and 835, 837 and 838, 840 and 841, 843 and 844, 846 and
847, 849 and 850, 852 and 853, 855 and 856, 858 and 859, 861 and
862, 864 and 865, 867 and 868, 870 and 871, 873 and 874, 876 and
877, 879 and 880, 882 and 883, 885 and 886, 888 and 889, 891 and
892, 894 and 895, 897 and 898, 900 and 901, 903 and 904, 906 and
907, 909 and 910, 912 and 913, 915 and 916, 918 and 919, 921 and
922, 924 and 925, 927 and 928, 930 and 931, 933 and 934, 936 and
937, 939 and 940, 942 and 943, 945 and 946, 948 and 949, 951 and
952, 954 and 955, 957 and 958, 960 and 961, 963 and 964, 966 and
967, 969 and 970, 972 and 973, 975 and 976, 978 and 979, 981 and
982, 984 and 985, 987 and 988, 990 and 991, 993 and 994, 996 and
997, 999 and 1000, 1002 and 1003, 1005 and 1006, 1008 and 1009,
1011 and 1012, 1014 and 1015, 1017 and 1018, 1020 and 1021, 1023
and 1024, 1026 and 1027, 1029 and 1030, 1032 and 1033, 1025 and
1036, 1038 and 1039, 1041 and 1042, 1044 and 1045, 1047 and 1048,
1050 and 1051, 1053 and 1054, 1056 and 1057, 1059 and 1060, 1062
and 1063, 1065 and 1066, 1068 and 1069, 1071 and 1072, 1074 and
1075, 1077 and 1078, 1080 and 1081, 1083 and 1084, 1086 and 1087,
1089 and 1090, 1092 and 1093, 1095 and 1096, 1098 and 1099, 1101
and 1102, 1104 and 1105, 1107 and 1108, 1110 and 1111, 1113 and
1114, 1116 and 1117, 1119 and 1120, 1122 and 1123, 1125 and 1126,
1128 and 1129, 1131 and 1132, 1134 and 1135, 1137 and 1138, 1140
and 1141, 1143 and 1144, 1146 and 1147, 1149 and 1150, 1152 and
1153, 1155 and 1156, 1158 and 1159, 1161 and 1162, 1164 and 1165,
1167 and 1168, 1170 and 1171, 1173 and 1174, 1176 and 1177, 1179
and 1180, 1182 and 1183, 1185 and 1186, 1188 and 1189, 1191 and
1192, 1194 and 1195, 1197 and 1198, 1200 and 1201, 1203 and 1204,
1206 and 1207, 1209 and 1210, 1212 and 1213, 1215 and 1216, 1218
and 1219, 1221 and 1222, 1224 and 1225, 1227 and 1228, 1230 and
1231, 1233 and 1234, 1236 and 1237, 1239 and 1240, 1242 and 1243,
1245 and 1246, 1248 and 1249, 1251 and 1252, 1254 and 1255, 1257
and 1258, 1260 and 1261, 1263 and 1264, 1266 and 1267, 1269 and
1270, 1272 and 1273, 1275 and 1276, 1278 and 1279, 1281 and 1282,
1284 and 1285, 1287 and 1288, 1290 and 1291, 1293 and 1294, 1296
and 1297, 1299 and 1300, 1302 and 1303, 1305 and 1306, 1308 and
1309, 1311 and 1312, 1314 and 1315, 1317 and 1318, 1320 and 1321,
1323 and 1324, 1326 and 1327, 1329 and 1330, 1332 and 1333, 1335
and 1336, 1338 and 1339, 1341 and 1342, 1344 and 1345, 1347 and
1348, 1350 and 1351, 1353 and 1354, 1356 and 1357, 1359 and 1360,
1362 and 1363, 1365 and 1366, 1368 and 1369, 1371 and 1372, 1374
and 1375, 1377 and 1378, 1380 and 1381, 1383 and 1384, 1386 and
1387, 1389 and 1390, 1392 and 1393, 1395 and 1396, 1398 and 1399,
1401 and 1402, 1404 and 1405, 1407 and 1408, 1410 and 1411, 1413
and 1414, 1416 and 1417, 1419 and 1420, 1422 and 1423, 1425 and
1426, 1428 and 1429, 1431 and 1432, 1434 and 1435, 1437 and 1438,
1440 and 1441, 1443 and 1444, 1446 and 1447, 1449 and 1450, 1452
and 1453, 1455 and 1456, 1458 and 1459, 1461 and 1462, 1464 and
1465, 1467 and 1468, 1470 and 1471, 1473 and 1474, 1476 and 1477,
1479 and 1480, 1482 and 1483, 1485 and 1486, 1488 and 1489, 1491
and 1492, 1494 and 1495, 1497 and 1498, 1500 and 1501, 1503 and
1504, 1506 and 1507, 1509 and 1510, 1512 and 1513, 1515 and 1516,
1518 and 1519, 1521 and 1522, 1524 and 1525, 1527 and 1528, 1530
and 1531, 1533 and 1534, 1536 and 1537, 1539 and 1540, 1542 and
1543, 1545 and 1546, 1548 and 1549, 1551 and 1552, 1554 and 1555,
1557 and 1558, 1560 and 1561, 1563 and 1564, 1566 and 1567, 1569
and 1570, 1572 and 1573, 1575 and 1576, 1578 and 1579, 1581 and
1582, 1584 and 15685, 1587 and 1588, 1590 and 1591, 1593 and 1594,
1596 and 1597, 1599 and 1600, 1602 and 1603, 1605 and 1606, 1608
and 1609, 1611 and 1612, 1614 and 1615, 1617 and 1618, 1620 and
1621, 1623 and 1624, 1626 and 1627, 1629 and 1630, 1632 and 1633,
1635 and 1636, 1638 and 1639, 1641 and 1642, 1644 and 1645, 1647
and 1648, 1650 and 1651, 1653 and 1654, 1656 and 1657, 1659 and
1660, 1662 and 1663, 1665 and 1666, 1668 and 1669, 1671 and 1672,
1674 and 1675, 1677 and 1678, 1680 and 1681, 1683 and 1684, 1686
and 1687, 1689 and 1690, 1692 and 1693, 1695 and 1696, 1698 and
1699, 1701 and 1702, 1704 and 1705, 1707 and 1708, 1710 and 1711,
1713 and 1714, 1716 and 1717, 1719 and 1720, 1722 and 1723, 1725
and 1726, 1728 and 1729, 1731 and 1732, 1734 and 1735, 1737 and
1738, 1740 and 1741, 1743 and 1744, 1746 and 1747, 1749 and 1750,
1752 and 1753, 1755 and 1756, 1758 and 1759, 1761 and 1762, 1764
and 1765, 1767 and 1768, 1770 and 1771, 1773 and 1774, 1776 and
1777, 1779 and 1780, 1782 and 1783, 1785 and 1786, 1788 and 1789,
1791 and 1792, 1794 and 1795, 1797 and 1798, 1800 and 1801, 1803
and 1804, 1806 and 1807, 1809 and 1810, 1812 and 1813, 1815 and
1816, 1818 and 1819, 1821 and 1822, 1824 and 1825, 1827 and 1828,
1830 and 1831, 1833 and 1834, 1836 and 1837, 1839 and 1840, 1842
and 1843, 1845 and 1846, 1848 and 1849, 1851 and 1852, 1854 and
1855, 1857 and 1858, 1860 and 1861, 1863 and 1864, 1866 and 1867,
1869 and 1870, 1872 and 1873, 1875 and 1876, 1878 and 1879, 1881
and 1882, 1884 and 1885, 1887 and 1888, 1890 and 1891, 1893 and
1894, 1896 and 1897, 1899 and 1900, 1902 and 1903, 1905 and 1906,
1908 and 1909, 1911 and 1912, 1914 and 1915, 1917 and 1918, 1920
and 1921, 1923 and 1924, 1926 and 1927, 1929 and 1930, 1932 and
1933, 1925 and 1936, 1938 and 1939, 1941 and 1942, 1944 and 1945,
1947 and 1948, 1950 and 1951, 1953 and 1954, 1956 and 157, 1959 and
1960, 1962 and 1963, 1965 and 1966, 1968 and 1969, 1971 and 1972,
1974 and 175, 1977 and 1978, 1980 and 1981, 1983 and 1984, 1986 and
1987, 1989 and 1999, 1992 and 1993, 1995 and 1996, 1998 and 1999,
2001 and 2002, 2004 and 2005, 2007 and 2008, 2010 and 2011, 2013
and 2014, 2016 and 2017, 2019 and 2020, 2022 and 2023, 2025 and
2026, 2028 and 2029, 2031 and 2032, 2034 and 2035, 2037 and 2038,
2040 and 2041, 2043 and 2044, 2046 and 2047, 2049 and 2050, 2052
and 2053, 2055 and 2056, or 2058 and 2059 of an NS3/4A nucleic acid
sequence such as SEQ ID NO: 1, an NS3/4A variant or consensus
nucleic acid sequence (e.g., an NS3/4A sequence developed from a
plurality of NS3/4A isotypes, such as SEQ ID NO: 35), or any NS3/4A
mutant (for example any nucleic acid encoding an NS3/4A with
altered protease activity). Preferably, the nucleic acids encoding
the inserted sequences retain the reading frame of the chimeric
NS3/4A polypeptide and when the TCE encodes an HCV TCE, the
sequence is inserted at a position that is not naturally occurring.
In some embodiments, the TCE is not an HCV peptide or peptide
fragment. In preferred embodiments, the nucleic acid encoding the
TCE can be inserted between nucleotides 1370 and 1548 of SEQ ID NO:
35, or in an analogous position in another NS3/4A coding sequence.
Aspects of the invention also include the polypeptides encoded by
the nucleic acid embodiments provided herein.
[0015] Accordingly, the nucleic acids encoding chimeric NS3/4A
polypeptides can encode an antigen, TCE, antigen and linker, TCE
and linker, antigen and adjuvant sequence, TCE and adjuvant
sequence, antigen and linker and adjuvant sequence, or TCE and
linker and adjuvant sequence inserted between amino acids 1 and 2,
2 and 3, 3 and 4, 4 and 5, 5 and 6, 6 and 7, 7 and 8, 8 and 9, 9
and 10, 10 and 11, 11 and 12, 12 and 13, 13 and 14, 14 and 15, 15
and 16, 16 and 17, 17 and 18, 18 and 19, 19 and 20, 20 and 21, 21
and 22, 22 and 23, 23 and 24, 24 and 25, 25 and 26, 26 and 27, 27
and 28, 28 and 29, 29 and 30, 30 and 31, 31 and 32, 32 and 33, 33
and 34, 34 and 35, 35 and 36, 36 and 37, 37 and 38, 38 and 39, 39
and 40, 40 and 41, 41 and 42, 42 and 43, 43 and 44, 44 and 45, 45
and 46, 46 and 47, 47 and 48, 48 and 49, 49 and 50, 50 and 51, 51
and 52, 52 and 53, 53 and 54, 54 and 55, 55 and 56, 56 and 57, 57
and 58, 58 and 59, 59 and 60, 60 and 61, 61 and 62, 62 and 63, 63
and 64, 64 and 65, 65 and 66, 66 and 67, 67 and 68, 68 and 69, 69
and 70, 70 and 71, 71 and 72, 72 and 73, 73 and 74, 74 and 75, 75
and 76, 76 and 77, 77 and 78, 78 and 79, 79 and 80, 80 and 81, 81
and 82, 82 and 83, 83 and 84, 84 and 85, 85 and 86, 86 and 87, 87
and 88, 88 and 89, 89 and 90, 90 and 91, 91 and 92, 92 and 93, 93
and 94, 94 and 95, 95 and 96, 96 and 97, 97 and 98, 98 and 99, 99
and 100, 100 and 101, 101 and 102, 102 and 103, 103 and 104, 104
and 105, 105 and 106, 106 and 107, 107 and 108, 108 and 109, 109
and 110, 110 and 111, 111 and 112, 112 and 113, 113 and 114, 114
and 115, 115 and 116, 116 and 117, 117 and 118, 118 and 119, 119
and 120, 120 and 121, 121 and 122, 122 and 123, 123 and 124, 124
and 125, 125 and 126, 126 and 127, 127 and 128, 128 and 129, 129
and 130, 130 and 131, 131 and 132, 132 and 133, 133 and 134, 134
and 135, 135 and 136, 136 and 137, 137 and 138, 138 and 139, 139
and 140, 140 and 141, 141 and 142, 142 and 143, 143 and 144, 144
and 145, 145 and 146, 146 and 147, 147 and 148, 148 and 149, 149
and 150, 150 and 151, 151 and 152, 152 and 153, 153 and 154, 154
and 155, 155 and 156, 156 and 157, 157 and 158, 158 and 159, 159
and 160, 160 and 161, 161 and 162, 162 and 163, 163 and 164, 164
and 165, 165 and 166, 166 and 167, 167 and 168, 168 and 169, 169
and 170, 170 and 171, 171 and 172, 172 and 173, 173 and 174, 174
and 175, 175 and 176, 176 and 177, 177 and 178, 178 and 179, 179
and 180, 180 and 181, 181 and 182, 182 and 183, 183 and 184, 184
and 185, 185 and 186, 186 and 187, 187 and 188, 188 and 189, 189
and 190, 190 and 191, 191 and 192, 192 and 193, 193 and 194, 194
and 195, 195 and 196, 196 and 197, 197 and 198, 198 and 199, 199
and 200, 200 and 201, 201 and 202, 202 and 203, 203 and 204, 204
and 205, 205 and 206, 206 and 207, 207 and 208, 208 and 209, 209
and 210, 210 and 211, 211 and 212, 212 and 213, 213 and 214, 214
and 215, 215 and 216, 216 and 217, 217 and 218, 218 and 219, 219
and 220, 220 and 221, 221 and 222, 222 and 223, 223 and 224, 224
and 225, 225, and 226, 226 and 227, 227 and 228, 228 and 229, 229
and 230, 230 and 231, 231 and 232, 232 and 233, 233 and 234, 234
and 235, 235 and 236, 236 and 237, 237 and 238, 238 and 239, 239
and 240, 240 and 241, 241 and 242, 242 and 243, 243 and 244, 244
and 245, 245 and 246, 246 and 247, 247 and 248, 248 and 249, 249
and 250, 250 and 251, 251 and 252, 252 and 253, 253 and 254, 254
and 255, 255 and 256, 256 and 257, 257 and 258, 258 and 259, 259
and 260, 260 and 261, 261 and 262, 262 and 263, 263 and 264, 264
and 265, 265 and 266, 266 and 267, 267 and 268, 268 and 269, 269
and 270, 270 and 271, 271 and 272, 272 and 273, 273 and 274, 274
and 275, 275 and 276, 276 and 277, 277 and 278, 278 and 279, 279
and 280, 280 and 281, 281 and 282, 282 and 283, 283 and 284, 284
and 285, 285 and 286, 286 and 287, 287 and 288, 288 and 289, 289
and 290, 290 and 291, 291 and 292, 292 and 293, 293 and 294, 294
and 295, 295 and 296, 296 and 297, 297 and 298, 298 and 299, 299
and 300, 300 and 201, 301 and 302, 302 and 303, 303 and 304, 304
and 305, 305 and 306, 306 and 307, 307 and 308, 308 and 309, 309
and 310, 310 and 311, 311 and 312, 312 and 313, 313 and 314, 314
and 315, 315 and 316, 316 and 317, 317 and 318, 318 and 319, 319
and 320, 320 and 321, 321, and 322, 322 and 323, 323 and 324, 324
and 325, 325, and 326, 326 and 327, 327 and 328, 328 and 329, 329
and 330, 330 and 331, 331 and 332, 332 and 333, 333 and 334, 334
and 335, 335 and 336, 336 and 337, 337 and 338, 338 and 339, 339
and 340, 340 and 341, 341 and 342, 342 and 343, 343 and 344, 344
and 345, 345 and 346, 346 and 347, 347 and 348, 348 and 349, 349
and 350, 350 and 351, 351 and 352, 352 and 353, 353 and 354, 354
and 355, 355 and 356, 356 and 357, 357 and 358, 358 and 359, 359
and 360, 360 and 361, 361 and 362, 362 and 363, 363 and 364, 364
and 365, 365 and 366, 366 and 367, 367 and 368, 368 and 369, 369
and 370, 370 and 371, 371 and 372, 372 and 373, 373 and 374, 374
and 375, 375 and 376, 376 and 377, 377 and 378, 378 and 379, 379
and 380, 380 and 381, 381 and 382, 382 and 383, 383 and 384, 384
and 385, 385 and 386, 386 and 387, 387 and 388, 388 and 389, 389
and 390, 390 and 391, 391 and 392, 392 and 393, 393 and 394, 394
and 395, 395 and 396, 396 and 397, 397 and 398, 398 and 399, 399
and 400, 401 and 402, 402 and 403, 403 and 404, 404 and 405, 405
and 406, 406 and 407, 407 and 408, 408 and 409, 409 and 410, 410
and 411, 411 and 412, 412 and 413, 413 and 414, 414 and 415, 415
and 416, 416 and 417, 417 and 418, 418 and 419, 419 and 420, 420
and 421, 421 and 422, 422 and 423, 423 and 424, 424 and 425, 425
and 426, 426 and 427, 427 and 428, 428 and 429, 429 and 430, 430
and 431, 431 and 432, 432 and 433, 433 and 434, 434 and 435, 435
and 436, 436 and 437, 437 and 438, 438 and 439, 439 and 440, 440
and 441, 441 and 442, 442 and 443, 443 and 444, 444 and 445, 445
and 446, 446 and 447, 447 and 448, 448 and 449, 449 and 450, 450
and 451, 451 and 452, 452 and 453, 453 and 454, 454 and 455, 455
and 456, 456 and 457, 457 and 458, 458 and 459, 459 and 460, 460
and 461, 461 and 462, 462 and 463, 463 and 464, 464 and 465, 465
and 466, 466 and 467, 467 and 468, 468 and 469, 469 and 470, 470
and 471, 471 and 472, 472 and 473, 473 and 474, 474 and 475, 475
and 476, 476 and 477, 477 and 478, 478 and 479, 479 and 480, 480
and 481, 481 and 482, 482 and 483, 483 and 484, 484 and 485, 485
and 486, 486 and 487, 487 and 488, 488 and 489, 489 and 490, 490
and 491, 491 and 492, 492 and 493, 493 and 494, 494 and 495, 495
and 496, 496 and 497, 497 and 498, 498 and 499, 499 and 500, 501
and 502, 502 and 503, 503 and 504, 504 and 505, 505 and 506, 506
and 507, 507 and 508, 508 and 509, 509 and 510, 510 and 511, 511
and 512, 512 and 513, 513 and 514, 514 and 515, 515 and 516, 516
and 517, 517 and 518, 518 and 519, 519 and 520, 520 and 521, 521
and 522, 522 and 523, 523 and 524, 524 and 525, 525 and 526, 526
and 527, 527 and 528, 528 and 529, 529 and 530, 530 and 531, 531
and 532, 532 and 533, 533 and 534, 534 and 535, 535 and 536, 536
and 537, 537 and 538, 538 and 539, 539 and 540, 540 and 541, 541
and 542, 542 and 543, 543 and 544, 544 and 545, 545 and 546, 546
and 547, 547 and 548, 548 and 549, 549 and 550, 550 and 551, 551
and 552, 552 and 553, 553 and 554, 554 and 555, 555 and 556, 556
and 557, 557 and 558, 558 and 559, 559 and 560, 560 and 561, 561
and 562, 562 and 563, 563 and 564, 564 and 565, 565 and 566, 566
and 567, 567 and 568, 568 and 569, 569 and 570, 570 and 571, 571
and 572, 572 and 573, 573 and 574, 574 and 575, 575 and 576, 576
and 577, 577 and 578, 578 and 579, 579 and 580, 580 and 581, 581
and 582, 582 and 583, 583 and 584, 584 and 585, 585 and 586, 586
and 587, 587 and 588, 588 and 589, 589 and 590, 590 and 591, 591
and 592, 592 and 593, 593 and 594, 594 and 595, 595 and 596, 596
and 597, 597 and 598, 598 and 599, 599 and 600, 601 and 602, 602
and 603, 603 and 604, 604 and 605, 605 and 606, 606 and 607, 607
and 608, 608 and 609, 609 and 610, 610 and 611, 611 and 612, 612
and 613, 613 and 614, 614 and 615, 615 and 616, 616 and 617, 617
and 618, 618 and 619, 619 and 620, 620 and 621, 621 and 622, 622
and 623, 623 and 624, 624 and 625, 625 and 626, 626 and 627, 627
and 628, 628 and 629, 629 and 630, 630 and 631, 631 and 632, 632
and 633, 633 and 634, 634 and 635, 635 and 636, 636 and 637, 637
and 638, 638 and 639, 639 and 640, 640 and 641, 641 and 642, 642
and 643, 643 and 644, 644 and 645, 645 and 646, 646 and 647, 647
and 648, 648 and 649, 649 and 650, 650 and 651, 651 and 652, 652
and 653, 653 and 654, 654 and 655, 655 and 656, 656 and 657, 657
and 658, 658 and 659, 659 and 660, 660 and 661, 661 and 662, 662
and 663, 663 and 664, 664 and 665, 665 and 666, 666 and 667, 667
and 668, 668 and 669, 669 and 670, 670 and 671, 671 and 672, 72 and
673, 673 and 674, 674 and 675, 675 and 676, 676 and 677, 677 and
678, 678 and 679, 679 and 680, 680 and 681, 681 and 682, 682 and
683, 683 and 684, 684 and 685, or 685 and 686 of an NS3/4A
polypeptide (e.g., SEQ ID NO: 2) an NS3/4A variant polypeptide
(e.g., SEQ ID NO: 36), or any NS3/4A mutant (for example any NS3/4A
with altered protease activity). In preferred embodiments, the
antigen, TCE, antigen and linker, TCE and linker, antigen and
adjuvant sequence, TCE and adjuvant sequence, antigen and linker
and adjuvant sequence, or TCE and linker and adjuvant sequence
inserted between amino acids 453 and 513 of SEQ ID NO: 36 or an
analogous position in an NS3/4A polypeptide. The encoded
polypeptides of the nucleic acid embodiments described herein are
also embodiments.
[0016] In some embodiments, the encoded antigen, TCE, antigen and
linker, TCE and linker, antigen and adjuvant sequence, TCE and
adjuvant sequence, antigen and linker and adjuvant sequence, or TCE
and linker and adjuvant sequence is juxtaposed to or flanking the
HCV NS3/4A polypeptide or fragment thereof. For example, the
nucleic acid encoding the antigen, TCE, antigen and linker, TCE and
linker, antigen and adjuvant sequence, TCE and adjuvant sequence,
antigen and linker and adjuvant sequence, or TCE and linker and
adjuvant sequence can be 5' to the nucleic acid encoding the HCV
NS3/4A polypeptide or fragment thereof, such that the TCE is on the
N-terminal end of the encoded chimeric polypeptide. Optionally, the
nucleic acid encoding the antigen, TCE, antigen and linker, TCE and
linker, antigen and adjuvant sequence, TCE and adjuvant sequence,
antigen and linker and adjuvant sequence, or TCE and linker and
adjuvant sequence can be 3' to the nucleic acid encoding the HCV
NS3/4A polypeptide or fragment thereof, such that the TCE is on the
C-terminal end of the encoded chimeric polypeptide.
[0017] The antigens and/or TCE used in the platforms described
herein can include, but are not limited to, nucleic acids that
encode viral antigens, bacterial antigens, parasitic antigens,
tumor antigens, and toxins. In some embodiments, the encoded TCE
comprises a sequence selected from the group consisting of SEQ ID
NOs: 221-571 and SEQ ID NOs: 809-1011 and SEQ ID NO: 1014 and SEQ
ID NOs: 1016-1034 and SEQ ID NOs: 1146-1173 and SEQ ID NOs:
1210-1328. In preferred embodiments, the encoded TCE or antigen is
obtained from a hepatitis virus, such as an antigen from the
Hepatitis A virus (HAV), Hepatitis B virus (HBV), or HCV, HIV, a
flu virus, an allergen (such as Birch allergen), or malaria. For
example, in some embodiments, the encoded TCE comprises the amino
acid sequence of SEQ ID NO: 1014. Antigens and TCEs that are
present on pathogens that infect domestic animals are also
embodied. That is, some embodiments include veterinary preparations
that comprise a nucleic acid encoding an NS3/4A platform, as
described herein, and an antigen present on an animal pathogen
(e.g., swine flu, avian flu, or equine flu).
[0018] Some embodiments also concern the chimeric polypeptides
encoded by the nucleic acids disclosed herein. In some embodiments,
the chimeric polypeptide encoded by the isolated nucleic acid
retains NS3A protease activity and/or NS3A helicase activity and/or
a protease cleavage site. Optionally, the chimeric polypeptides
include a linker or adjuvant polypeptide (e.g., an RNA binding
domain, such as poly Arg). For example, in some embodiments, the
linker flanks at least one end (i.e., N-terminal or C-terminal end)
of the TCE. In some embodiments, a linker comprising one to six
alanine and/or glycine residues is flanking or juxtaposed to at
least one end of the TCE, such as between NS3/4A amino acid
sequences and TCE sequences.
[0019] Also provided herein are NS3/4A chimeric polypeptides that
include NS3/4A polypeptides or variants, or fragments thereof and
at least one antigen, TCE, antigen and linker, TCE and linker,
antigen and adjuvant sequence, TCE and adjuvant sequence, antigen
and linker and adjuvant sequence, or TCE and linker and adjuvant
sequence. Optionally, these sequences are inserted within the HCV
NS3/4A polypeptide or fragment thereof. For example, the chimeric
NS3/4A polypeptides can consist of, consist essentially of, or
comprise an antigen, TCE, antigen and linker, TCE and linker,
antigen and adjuvant sequence, TCE and adjuvant sequence, antigen
and linker and adjuvant sequence, or TCE and linker and adjuvant
sequence inserted between amino acids 1 and 2, 2 and 3, 3 and 4, 4
and 5, 5 and 6, 6 and 7, 7 and 8, 8 and 9, 9 and 10, 10 and 11, 11
and 12, 12 and 13, 13 and 14, 14 and 15, 15 and 16, 16 and 17, 17
and 18, 18 and 19, 19 and 20, 20 and 21, 21 and 22, 22 and 23, 23
and 24, 24 and 25, 25 and 26, 26 and 27, 27 and 28, 28 and 29, 29
and 30, 30 and 31, 31 and 32, 32 and 33, 33 and 34, 34 and 35, 35
and 36, 36 and 37, 37 and 38, 38 and 39, 39 and 40, 40 and 41, 41
and 42, 42 and 43, 43 and 44, 44 and 45, 45 and 46, 46 and 47, 47
and 48, 48 and 49, 49 and 50, 50 and 51, 51 and 52, 52 and 53, 53
and 54, 54 and 55, 55 and 56, 56 and 57, 57 and 58, 58 and 59, 59
and 60, 60 and 61, 61 and 62, 62 and 63, 63 and 64, 64 and 65, 65
and 66, 66 and 67, 67 and 68, 68 and 69, 69 and 70, 70 and 71, 71
and 72, 72 and 73, 73 and 74, 74 and 75, 75 and 76, 76 and 77, 77
and 78, 78 and 79, 79 and 80, 80 and 81, 81 and 82, 82 and 83, 83
and 84, 84 and 85, 85 and 86, 86 and 87, 87 and 88, 88 and 89, 89
and 90, 90 and 91, 91 and 92, 92 and 93, 93 and 94, 94 and 95, 95
and 96, 96 and 97, 97 and 98, 98 and 99, 99 and 100, 100 and 101,
101 and 102, 102 and 103, 103 and 104, 104 and 105, 105 and 106,
106 and 107, 107 and 108, 108 and 109, 109 and 110, 110 and 111,
111 and 112, 112 and 113, 113 and 114, 114 and 115, 115 and 116,
116 and 117, 117 and 118, 118 and 119, 119 and 120, 120 and 121,
121 and 122, 122 and 123, 123 and 124, 124 and 125, 125 and 126,
126 and 127, 127 and 128, 128 and 129, 129 and 130, 130 and 131,
131 and 132, 132 and 133, 133 and 134, 134 and 135, 135 and 136,
136 and 137, 137 and 138, 138 and 139, 139 and 140, 140 and 141,
141 and 142, 142 and 143, 143 and 144, 144 and 145, 145 and 146,
146 and 147, 147 and 148, 148 and 149, 149 and 150, 150 and 151,
151 and 152, 152 and 153, 153 and 154, 154 and 155, 155 and 156,
156 and 157, 157 and 158, 158 and 159, 159 and 160, 160 and 161,
161 and 162, 162 and 163, 163 and 164, 164 and 165, 165 and 166,
166 and 167, 167 and 168, 168 and 169, 169 and 170, 170 and 171,
171 and 172, 172 and 173, 173 and 174, 174 and 175, 175 and 176,
176 and 177, 177 and 178, 178 and 179, 179 and 180, 180 and 181,
181 and 182, 182 and 183, 183 and 184, 184 and 185, 185 and 186,
186 and 187, 187 and 188, 188 and 189, 189 and 190, 190 and 191,
191 and 192, 192 and 193, 193 and 194, 194 and 195, 195 and 196,
196 and 197, 197 and 198, 198 and 199, 199 and 200, 200 and 201,
201 and 202, 202 and 203, 203 and 204, 204 and 205, 205 and 206,
206 and 207, 207 and 208, 208 and 209, 209 and 210, 210 and 211,
211 and 212, 212 and 213, 213 and 214, 214 and 215, 215 and 216,
216 and 217, 217 and 218, 218 and 219, 219 and 220, 220 and 221,
221 and 222, 222 and 223, 223 and 224, 224 and 225, 225, and 226,
226 and 227, 227 and 228, 228 and 229, 229 and 230, 230 and 231,
231 and 232, 232 and 233, 233 and 234, 234 and 235, 235 and 236,
236 and 237, 237 and 238, 238 and 239, 239 and 240, 240 and 241,
241 and 242, 242 and 243, 243 and 244, 244 and 245, 245 and 246,
246 and 247, 247 and 248, 248 and 249, 249 and 250, 250 and 251,
251 and 252, 252 and 253, 253 and 254, 254 and 255, 255 and 256,
256 and 257, 257 and 258, 258 and 259, 259 and 260, 260 and 261,
261 and 262, 262 and 263, 263 and 264, 264 and 265, 265 and 266,
266 and 267, 267 and 268, 268 and 269, 269 and 270, 270 and 271,
271 and 272, 272 and 273, 273 and 274, 274 and 275, 275 and 276,
276 and 277, 277 and 278, 278 and 279, 279 and 280, 280 and 281,
281 and 282, 282 and 283, 283 and 284, 284 and 285, 285 and 286,
286 and 287, 287 and 288, 288 and 289, 289 and 290, 290 and 291,
291 and 292, 292 and 293, 293 and 294, 294 and 295, 295 and 296,
296 and 297, 297 and 298, 298 and 299, 299 and 300, 300 and 201,
301 and 302, 302 and 303, 303 and 304, 304 and 305, 305 and 306,
306 and 307, 307 and 308, 308 and 309, 309 and 310, 310 and 311,
311 and 312, 312 and 313, 313 and 314, 314 and 315, 315 and 316,
316 and 317, 317 and 318, 318 and 319, 319 and 320, 320 and 321,
321, and 322, 322 and 323, 323 and 324, 324 and 325, 325, and 326,
326 and 327, 327 and 328, 328 and 329, 329 and 330, 330 and 331,
331 and 332, 332 and 333, 333 and 334, 334 and 335, 335 and 336,
336 and 337, 337 and 338, 338 and 339, 339 and 340, 340 and 341,
341 and 342, 342 and 343, 343 and 344, 344 and 345, 345 and 346,
346 and 347, 347 and 348, 348 and 349, 349 and 350, 350 and 351,
351 and 352, 352 and 353, 353 and 354, 354 and 355, 355 and 356,
356 and 357, 357 and 358, 358 and 359, 359 and 360, 360 and 361,
361 and 362, 362 and 363, 363 and 364, 364 and 365, 365 and 366,
366 and 367, 367 and 368, 368 and 369, 369 and 370, 370 and 371,
371 and 372, 372 and 373, 373 and 374, 374 and 375, 375 and 376,
376 and 377, 377 and 378, 378 and 379, 379 and 380, 380 and 381,
381 and 382, 382 and 383, 383 and 384, 384 and 385, 385 and 386,
386 and 387, 387 and 388, 388 and 389, 389 and 390, 390 and 391,
391 and 392, 392 and 393, 393 and 394, 394 and 395, 395 and 396,
396 and 397, 397 and 398, 398 and 399, 399 and 400, 401 and 402,
402 and 403, 403 and 404, 404 and 405, 405 and 406, 406 and 407,
407 and 408, 408 and 409, 409 and 410, 410 and 411, 411 and 412,
412 and 413, 413 and 414, 414 and 415, 415 and 416, 416 and 417,
417 and 418, 418 and 419, 419 and 420, 420 and 421, 421 and 422,
422 and 423, 423 and 424, 424 and 425, 425 and 426, 426 and 427,
427 and 428, 428 and 429, 429 and 430, 430 and 431, 431 and 432,
432 and 433, 433 and 434, 434 and 435, 435 and 436, 436 and 437,
437 and 438, 438 and 439, 439 and 440, 440 and 441, 441 and 442,
442 and 443, 443 and 444, 444 and 445, 445 and 446, 446 and 447,
447 and 448, 448 and 449, 449 and 450, 450 and 451, 451 and 452,
452 and 453, 453 and 454, 454 and 455, 455 and 456, 456 and 457,
457 and 458, 458 and 459, 459 and 460, 460 and 461, 461 and 462,
462 and 463, 463 and 464, 464 and 465, 465 and 466, 466 and 467,
467 and 468, 468 and 469, 469 and 470, 470 and 471, 471 and 472,
472 and 473, 473 and 474, 474 and 475, 475 and 476, 476 and 477,
477 and 478, 478 and 479, 479 and 480, 480 and 481, 481 and 482,
482 and 483, 483 and 484, 484 and 485, 485 and 486, 486 and 487,
487 and 488, 488 and 489, 489 and 490, 490 and 491, 491 and 492,
492 and 493, 493 and 494, 494 and 495, 495 and 496, 496 and 497,
497 and 498, 498 and 499, 499 and 500, 501 and 502, 502 and 503,
503 and 504, 504 and 505, 505 and 506, 506 and 507, 507 and 508,
508 and 509, 509 and 510, 510 and 511, 511 and 512, 512 and 513,
513 and 514, 514 and 515, 515 and 516, 516 and 517, 517 and 518,
518 and 519, 519 and 520, 520 and 521, 521 and 522, 522 and 523,
523 and 524, 524 and 525, 525 and 526, 526 and 527, 527 and 528,
528 and 529, 529 and 530, 530 and 531, 531 and 532, 532 and 533,
533 and 534, 534 and 535, 535 and 536, 536 and 537, 537 and 538,
538 and 539, 539 and 540, 540 and 541, 541 and 542, 542 and 543,
543 and 544, 544 and 545, 545 and 546, 546 and 547, 547 and 548,
548 and 549, 549 and 550, 550 and 551, 551 and 552, 552 and 553,
553 and 554, 554 and 555, 555 and 556, 556 and 557, 557 and 558,
558 and 559, 559 and 560, 560 and 561, 561 and 562, 562 and 563,
563 and 564, 564 and 565, 565 and 566, 566 and 567, 567 and 568,
568 and 569, 569 and 570, 570 and 571, 571 and 572, 572 and 573,
573 and 574, 574 and 575, 575 and 576, 576 and 577, 577 and 578,
578 and 579, 579 and 580, 580 and 581, 581 and 582, 582 and 583,
583 and 584, 584 and 585, 585 and 586, 586 and 587, 587 and 588,
588 and 589, 589 and 590, 590 and 591, 591 and 592, 592 and 593,
593 and 594, 594 and 595, 595 and 596, 596 and 597, 597 and 598,
598 and 599, 599 and 600, 601 and 602, 602 and 603, 603 and 604,
604 and 605, 605 and 606, 606 and 607, 607 and 608, 608 and 609,
609 and 610, 610 and 611, 611 and 612, 612 and 613, 613 and 614,
614 and 615, 615 and 616, 616 and 617, 617 and 618, 618 and 619,
619 and 620, 620 and 621, 621 and 622, 622 and 623, 623 and 624,
624 and 625, 625 and 626, 626 and 627, 627 and 628, 628 and 629,
629 and 630, 630 and 631, 631 and 632, 632 and 633, 633 and 634,
634 and 635, 635 and 636, 636 and 637, 637 and 638, 638 and 639,
639 and 640, 640 and 641, 641 and 642, 642 and 643, 643 and 644,
644 and 645, 645 and 646, 646 and 647, 647 and 648, 648 and 649,
649 and 650, 650 and 651, 651 and 652, 652 and 653, 653 and 654,
654 and 655, 655 and 656, 656 and 657, 657 and 658, 658 and 659,
659 and 660, 660 and 661, 661 and 662, 662 and 663, 663 and 664,
664 and 665, 665 and 666, 666 and 667, 667 and 668, 668 and 669,
669 and 670, 670 and 671, 671 and 672, 72 and 673, 673 and 674, 674
and 675, 675 and 676, 676 and 677, 677 and 678, 678 and 679, 679
and 680, 680 and 681, 681 and 682, 682 and 683, 683 and 684, or 684
and 685 of an NS3/4A polypeptide (e.g., SEQ ID NO: 2) or NS3/4A
variant polypeptide, (e.g., SEQ ID NO: 36), or fragment thereof. In
preferred embodiments, the chimeric NS3/4A polypeptides can consist
of, consist essentially of, or comprise an antigen, TCE, antigen
and linker, TCE and linker, antigen and adjuvant sequence, TCE and
adjuvant sequence, antigen and linker and adjuvant sequence, or TCE
and linker and adjuvant sequence inserted between amino acids 453
and 513 of SEQ ID NO: 36, or in an analogous position in any NS3/4A
polypeptide.
[0020] In some embodiments, the chimeric NS3/4A polypeptide
includes an antigen, TCE, antigen and linker, TCE and linker,
antigen and adjuvant sequence, TCE and adjuvant sequence, antigen
and linker and adjuvant sequence, or TCE and linker and adjuvant
sequence that is juxtaposed to or flanking the NS3/4A polypeptide
or a fragment thereof. For example, the TCE can be juxtaposed to or
flanking the N-terminal or C-terminal end of the chimeric NS3/4A
polypeptide or NS3/4A variant. In preferred embodiments, the
chimeric NS3/4A polypeptides retain NS3 protease activity, and/or
NS3 helicase activity.
[0021] In some embodiments, the TCE can be derived from antigens
such as viral antigens, bacterial antigens, parasitic antigens,
tumor antigens, allergens and toxins. For example, in some
embodiments, the TCE comprises a sequence selected from the group
consisting of SEQ ID NOs: 221-571 and SEQ ID NOs: 809-1011 and SEQ
ID NO: 1014 and SEQ ID NOs: 1016-1034 and SEQ ID NOs: 1146-1173 and
SEQ ID NOs: 1210-1328. In preferred embodiments, the encoded TCE or
antigen is obtained from a hepatitis virus, such as an antigen from
the Hepatitis A virus (HAV), Hepatitis B virus (HBV), or HCV or
HIV, flu, Birch allergens or malaria. For example, in some
embodiments, the TCE comprises the amino acid sequence of SEQ ID
NO: 1014. Antigens and TCEs that are present on pathogens that
infect domestic animals are also embodied. That is, some
embodiments include veterinary preparations that comprise a NS3/4A
platform, as described herein, and an antigen present on an animal
pathogen (e.g., swine flu, avian flu, or equine flu).
[0022] Another embodiment disclosed herein includes a composition
that comprises a recombinant peptide immunogen comprising at least
one antigen and a hepatitis C virus (HCV) NS3 protease cleavage
site, wherein the HCV NS3 protease cleavage site is joined to the
antigen at a position that is not naturally occurring. In some
embodiments, the antigen comprises an epitope from a plant, virus,
bacteria, or a cancer cell. In other embodiments, the antigen is
not a peptide from HCV. In still other embodiments, the antigenic
fragment comprises an epitope from birch, peanut, wheat protein, a
hepatitis viral protein, a hepatitis B viral protein, or hepatitis
B virus core protein (HBcAg). In other embodiments, the antigenic
fragment comprises a fragment of an antigen presented in SEQ ID
NOs: 1016-1034, SEQ ID NOs: 1146-1173 and SEQ ID NOs:
1210-1328.
[0023] In some aspects, the recombinant peptide immunogen comprises
a plurality of NS3 protease cleavage sites. In some embodiments,
the NS3 protease cleavage site is chosen from the group consisting
of NS3/4A, NS4A/B, NS4B/5A, and NS5A/B. In some embodiments, the
HCV NS3 protease cleavage site comprises the sequence:
SADLEVVTSTWVLVGGVL (SEQ ID NO: 1340). In other embodiments, the HCV
NS3 protease cleavage site comprises the sequence: DEMEECSQHLPYIEQG
(SEQ ID NO: 1341). In still other embodiments, the HCV NS3 protease
cleavage site comprises a sequence from the sequences presented
below:
TABLE-US-00001 HCV Strain SEQ name: Amino acid sequence: Junction
ID: H-FDA CMSADLEVVT STWVLVGGVL N53/4A 1342 H-AP CMSADLEVVT
STWVLVGGVL N53/4A 1343 HCV-1 CMSADLEVVT STWVLVGGVL N53/4A 1344
HCV-J CMSADLEVVT STWVLVGGVL N53/4A 1345 HCV-BK CMSADLEVVT
STWVLVGGVL N53/4A 1346 HC-J6 CMQADLEVMT STWVLAGGVL N53/4A 1347
HCV-T CMSADLEVVT STWVLVGGVL NS3/4A 1348 HC-J8 CMQADLEIMT SSWVLAGGVL
NS3/4A 1349 HCV-JT,JT CMSAQLEVVT STWVLVGGVL NS3/4A 1350 H-FDA
YQEFDEMEEC SQHLPYIEQG NS4A/4B 1351 H-AP YQEFDEMEEC SQHLPYIEQG
NS4A/4B 1352 HCV-1 YREFDEMEEC SQHLPYIEQG NS4A/4B 1353 HCV-J
YQEFDEMEEC ASHLPYIEQG NS4A/4B 1354 HCV-BK YQEFDEMEEC ASHLPYIEQG
NS4A/4B 1355 HC-J1,4 YEAFDEMEEC ASRAALIEEG NS4A/4B 1356 HCV-T
YQEFDEMEEC ASHLPYIEQG NS4A/4B 1357 HC-J8 YQAFDEMEEC ASKAALIEEG
NS4A/4B 1358 HCV-JT,JT' YREFDEMEEC ASHLPYIEQG NS4A/4B 1359 H-FDA
WISSECTTPC SGSWLRDIWD NS4B/5A 1360 H-AP WISSECTTPC SGSWLRDIWD
NS4B/5A 1361 HCV-1 WISSECTTPC SGSWLRDIWD NS4B/5A 1362 HCV-J
WINEDCSTPC SGSWLKDVWD NS4B/5A 1363 HCV-BK WINEDCSTPC SGSWLRDVWD
NS4B/5A 1364 HC-J6 WITEDCPIPC SGSWLRDVWD NS4B/5A 1365 HCV-T
WINEDCSTPC SGSWLRDVWD NS4B/5A 1366 HC-J8 WITEDCPVPC SGSWLQDIWD
NS4B/5A 1367 HCV-JT WINEDCSTPC SGSWLKDVWD NS4B/5A 1368 HCV-JT'
WINEDCSTPC SGSWLRDVWD NS4B/5A 1369 H-FDA GADTEDVVCC SMSYTWTGAL
NS5A/5B 1370 H-AP GADTEDVVCC SMSYSWTGAL NS5A/5B 1371 HCV-1
EANAEDVVCC SMSYSWTGAL NS5A/5B 1372 HCV-J GEAGEDVVCC SMSYTWTGAL
NS5A/5B 1373 HCV-BK EEASEDVVCC SMSYTWTGAL NS5A/5B 1374 HC-J6
SEEDDSVVCC SMSYSWTGAL NS5A/5B 1375 HCV-T EEDGEGVICC SMSYTWTGAL
NS5A/5B 1376 HC-J8 SDQEDSVICC SMSYSWTGAL NS5A/5B 1377 HCV-JT,JT'
GEASDDIVCC SMSYTWTGAL NS5A/5B 1378 CONSENSUS D C S CONSENSUS E T
A
[0024] In other aspects, the recombinant peptide immunogen
comprises a plurality of antigenic fragments of a protein assembled
in a non-naturally occurring order. In other aspects, the
recombinant peptide immunogen comprises a plurality of antigenic
fragments of a protein assembled in a naturally occurring order. In
still other aspects, the recombinant peptide immunogen comprises a
plurality of antigenic fragments from at least two different
proteins assembled in a non-naturally occurring order.
[0025] In more aspects, the composition further comprises an NS3/4A
peptide. In some embodiments, the NS3/4A peptide comprises a
mutation that enhances protease activity. In some embodiments, the
mutation is selected from the group consisting of Tyr6Ala,
Arg11Ala, Leu13Ala, Leu14Ala, Glu30Ala, Cys52Ala, Gly58Ala,
Ala59Gly, Ile64Ala, Ile64Ala, Gln73Ala, Thr76Ala, Pro86Ala,
Ala111Gly, Gly 122Ala, Tyr 134Ala, Lys 136Ala, Gly 141Ala,
Val158Ala, Arg161Ala, Ala166Gly, and Thr177Ala. In some
embodiments, the NS3/4A peptide is joined to the peptide immunogen.
In some embodiments, the NS3/4A peptide is C-terminal with respect
to the peptide immunogen. In other embodiments, the NS3/4A peptide
is N-terminal with respect to the peptide immunogen. In still other
embodiments, the NS3/4A peptide is inserted within the peptide
immunogen. In other embodiments, the peptide immunogen is inserted
within the NS3/4A peptide. In yet other embodiments, the NS3/4A
peptide is not joined to the peptide immunogen.
[0026] Another embodiment disclosed herein includes a composition
that comprises a nucleic acid encoding a recombinant peptide
immunogen comprising an antigen and a hepatitis C virus (HCV) NS3
protease cleavage site, wherein the HCV NS3 protease cleavage site
is joined to the antigen at a position that is not naturally
occurring. In some embodiments, the antigen comprises an epitope
from a plant, virus, bacteria, or cancer cell. In some embodiments,
the antigen is not a peptide from HCV. In some embodiments, the
antigen is an antigenic fragment of a birch, peanut, wheat protein,
an antigenic fragment of a hepatitis viral protein, an antigenic
fragment of a hepatitis B virus protein, an antigenic fragment of
the hepatitis B virus core protein (HBcAg).
[0027] In some aspects, the recombinant peptide immunogen comprises
a plurality of NS3 protease cleavage sites. In some embodiments,
the NS3 protease cleavage site is chosen from the group consisting
of NS3/4A, NS4A/B, NS4B/5A, and NS5A/B. In some embodiments, the
HCV NS3 protease cleavage site comprises the sequence:
SADLEVVTSTWVLVGGVL (SEQ ID NO: 1340). In other embodiments, the HCV
NS3 protease cleavage site comprises the sequence: DMEECSQHLPYIEQG
(SEQ ID NO: 1341).
[0028] In other aspects, the recombinant peptide immunogen
comprises a plurality of antigenic fragments of a protein assembled
in a non-naturally occurring order. In other aspects, the
recombinant peptide immunogen comprises a plurality of antigenic
fragments of a protein assembled in a naturally occurring order. In
still other aspects, the recombinant peptide immunogen comprises a
plurality of antigenic fragments from at least two different
proteins assembled in a non-naturally occurring order. In some
embodiments, a peptide immunogen described herein is not native to
hepatitis C. In other embodiments, a peptide immunogen described
herein is not native to a hepatitis virus. In other embodiments, a
peptide immunogen described herein is not native to influenza.
[0029] In some aspects, the composition further comprises a nucleic
acid encoding NS3/4A peptide. In some embodiments, the nucleic acid
coding for NS3/4A peptide is codon-optimized for expression in a
mammal or bird. In other embodiments, the nucleic acid coding the
NS3/4A peptide is codon-optimized for expression in a human, dog,
cat, horse, pig, cow, goat, or chicken In some embodiments, the
NS3/4A peptide comprises a mutation that enhances protease
activity. In some embodiments, the mutation is selected from the
group consisting of Tyr6Ala, Arg11Ala, Leu13Ala, Leu14Ala,
Glu30Ala, Cys52Ala, Gly58Ala, Ala59Gly, Ile64Ala, Ile64Ala,
Gln73Ala, Thr76Ala, Pro86Ala, Ala111Gly, Gly 122Ala, Tyr 134Ala,
Lys 136Ala, Gly 141Ala, Val158Ala, Arg161Ala, Ala166Gly, and
Thr177Ala. In some embodiments, the nucleic acid encoding NS3/4A
peptide is joined to the nucleic acid encoding the peptide
immunogen. In yet other embodiments, the nucleic acid encoding
NS3/4A peptide is not joined to the peptide immunogen.
[0030] In an alternative aspect, the composition further comprises
the NS3/4A peptide. In some embodiments, the NS3/4A peptide
comprises a mutation that enhances protease activity. In some
embodiments, the mutation is selected from the group consisting of
Tyr6Ala, Arg11Ala, Leu13Ala, Leu14Ala, Glu30Ala, Cys52Ala,
Gly58Ala, Ala59Gly, Ile64Ala, Ile64Ala, Gln73Ala, Thr76Ala,
Pro86Ala, Ala111Gly, Gly 122Ala, Tyr 134Ala, Lys 136Ala, Gly
141Ala, Val158Ala, Arg161Ala, Ala166Gly, and Thr177Ala. In some
embodiment, the NS3/4A peptide is joined to the peptide immunogen.
In some embodiments, the NS3/4A peptide is C-terminal with respect
to the peptide immunogen. In other embodiments, the NS3/4A peptide
is N-terminal with respect to the peptide immunogen. In still other
embodiments, the NS3/4A peptide is inserted within the peptide
immunogen. In other embodiments, the peptide immunogen is inserted
within the NS3/4A peptide. In yet other embodiments, the NS3/4A
peptide is not joined to the peptide immunogen.
[0031] It has been discovered that certain mutations in the NS3
domain of HCV allow for protease cleavage of some substrates but
not others. More particularly, it was found that alanine
substitution at positions 1050 and 1060 of the NS3 protease created
protease molecules with altered substrate specificity. The HCV NS3
mutants V1050A and Q1060A were found to cleave at the NS3-NS4A
junction but were unable to cleave the cellular substrate
IPS-1/Cardif/MAVS/VISA. Since cleavage of IPS-1/Cardif/MAVS/VISA by
wild-type NS3 reduces the response to the toll-like receptors
(TLR-3) and the RIG-1 pathway, which in turn impairs interferon
(IFN) alpha and beta signaling, it is contemplated that the HCV NS3
V1055A and Q1060A mutants allow for cleavage of the NS3-4A junction
without reducing interferon alpha and beta signaling. Type I
interferons are central mediators for antiviral responses.
Interferon-promoter stimulator 1 (IPS-1) contains an N-terminal
CARD-like structure that mediates interaction with the CARD of
RIG-I and Mda5, which are cytoplasmic RNA helicases that sense
viral infection. `Knockdown` of IPS-1 by small interfering RNA
blocks interferon induction by virus infection. Thus, IPS-1 is an
adaptor involved in RIG-I- and Mda5-mediated antiviral immune
responses.
[0032] Accordingly, HCV NS3 mutants V1055A and Q1060A, protease
active fragments of these molecules containing said mutations, and
nucleic acids encoding these molecules are therefore useful in the
HCV immunogen fusion proteins or vaccines described herein for
induction of an immune response against HCV when elevated
interferon alpha and beta signaling (e.g., amounts of IFN-alpha or
IFN-beta commensurate with amounts of IFN-alpha or IFN-beta in
uninfected cells or an uninfected individual) is desired.
Additionally, the HCV NS3 mutants V1050A and Q1060A, protease
active fragments of these molecules containing said mutations, and
nucleic acids encoding these molecules are useful as platforms for
incorporation or attachment of heterologous peptides to which an
immune response is desired when elevated interferon alpha and beta
signaling (e.g., amounts of IFN-alpha or IFN-beta commensurate with
amounts of IFN-alpha or IFN-beta in uninfected cells or an
uninfected individual) is preferred. Furthermore, HCV NS3 mutants
V1050A and Q1060A, protease active fragments of these molecules
containing said mutations, and nucleic acids encoding these
molecules can be provided in combination with other immunogenic
peptides or nucleic acids encoding immunogenic peptides so as
induce an adjuvant activity toward said immunogenic peptides or to
otherwise induce an immune response characterized by interferon
alpha and beta signaling commensurate with that observed in
uninfected cells or an uninfected individual.
[0033] Embodiments comprise, consist, or consist essentially of the
peptides (SEQ. ID. NOS.: 68, 73, and 1329) or fragments thereof
that retain protease activity, nucleic acids encoding these
molecules, vectors having said nucleic acids, and cells having said
vectors, nucleic acids, or peptides. Additional embodiments include
an NS3 or NS3/4A encoding nucleic acid or fragment thereof or
corresponding peptide, which comprise a sequence that was optimized
for codons most frequently used in humans. That is, more
embodiments comprise, consist, or consist essentially of nucleic
acids that have been codon optimized for expression in humans,
which encode the mutant HCV peptides described herein (e.g., SEQ.
ID. NOS.: 68, 73, and 1329) or fragments thereof that retain
protease activity. Vectors having said codon-optimized nucleic
acids, immunogenic preparations, and vaccines having said
codon-optimized nucleic acids and vectors and cells having said
vectors are also embodiments. Preferred embodiments include DNA
immunogens that comprise, consist, or consist essentially of
nucleic acids encoding SEQ. ID. NOS 68, 73, and 1329 or a fragment
thereof that retain protease activity. These DNA immunogens can be
provided to cells by transfection, injection, electroporation,
needle electroporation (e.g., Medpulsar or Elgin), gene gun,
transdermal application, or intranasal application.
[0034] In one embodiment, specific mutations of the NS3 protease
domain can be made and screened to find specific mutants that have
little effect, no effect, or heightened effect on protease cleavage
at the NS3-NS4a cleavage site while losing the ability to cleave
IPS-1 to .DELTA.IPS-1. In a preferred embodiment, amino acids 1053
through 1062 of the NS3/4A gene, corresponding to amino acids 27
through 36 of SEQ ID NOs.: 1330-1339 have been identified as
affecting the specificity of the NS3 protease domain. Accordingly,
mutations in this region of the NS3/4A or fragments containing
mutations in this region can be selected for their ability to
cleave the NS3-NS4A cleavage site while being unable to cleave
IPS-1 to .DELTA.IPS-1.
[0035] A large scale mutational analysis of HCV NS3 domain was
undertaken. This project produced a variety of truncated versions
of the NS3/4A peptide (e.g., SEQ. ID. NOs.: 12 and 13) and mutants
that lacked a proteolytic cleavage site (e.g., SEQ. ID. NOs.:
3-11). Other HCV NS3 mutants were found to have altered protease
activity (e.g., SEQ ID NOs: 40-220). For example, some mutants,
which have an alanine substitution or a glycine substitution in the
NS3 protease domain were found to have an abolished, reduced,
enhanced, or greatly enhanced protease activity. Exemplary mutants
include SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43,
SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID
NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52,
SEQ ID NO; 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID
NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO; 61,
SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID
NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70,
SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID
NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79,
SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID
NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88,
SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID
NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97,
SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ
ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID
NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO:
110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO:
114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO:
118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO:
122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO:
126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO:
130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO:
134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO:
138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO:
142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO:
146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO:
150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO:
154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO:
158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO:
162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO:
166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO:
170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO:
174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO:
178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO:
182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO:
186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO:
190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO:
194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO:
198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO:
202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO:
206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO:
210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO:
214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO:
218, SEQ ID NO: 219, or SEQ ID NO: 220. Accordingly, regions of the
NS3 domain, which retained protease activity was carefully mapped
by mutational analysis. Surprisingly, approximately 87% of the
protease residues could be replaced and protease activity was
retained.
[0036] Aspects of the present invention include compositions that
comprise, consist, or consist essentially of the nucleic acid
sequence provided by the sequence of SEQ. ID. NO.: 35 and/or the
peptide sequence provided by the sequence of SEQ. ID. NO.: 36.
Preferred embodiments, for example, include compositions that
comprise, consist or consist essentially of any number of
consecutive nucleotides between at least 12-2112 nucleotides of
SEQ. ID. NO.: 35 or a complement thereof (e.g., 12-15, 15-20,
20-30, 30-50, 50-100, 100-200, 200-500, 500-1000, 1000-1500,
1500-2079, or 1500-2112 consecutive nucleotides). Preferred
embodiments also include compositions that comprise, consist or
consist essentially of any number of consecutive nucleotides
between at least 12-2112 nucleotides of SEQ. ID. NO.: 35 or a
complement thereof (e.g., at least 3, 4, 6, 8, 10, 12, 15, 20, 30,
40, 50, 60, 70, 80, 90, or 100 consecutive nucleotides acids of
SEQ. ID. NO.: 35). Additional embodiments include nucleic acids
that comprise, consist, or consist essentially of a sequence that
encodes SEQ. ID. NO.: 36 or a fragment thereof, that is, any number
of consecutive amino acids between at least 3-50 amino acids of
SEQ. ID. NO.: 36 (e.g., 3, 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40,
45, or 50 consecutive amino acids). Still more embodiments include
peptides that comprises, consist, or consist essentially of the
sequence of SEQ. ID. NO.: 36 or a fragment thereof, that is, any
number of consecutive amino acids between at least 3-50 amino acids
of SEQ. ID. NO.: 36 (e.g., 3, 4, 6, 8, 10, 12, 15, 20, 25, 30, 35,
40, 45, or 50 consecutive amino acids).
[0037] Other preferred embodiments include compositions that
comprise, consist, or consist essentially of any number of
consecutive nucleotides between at least 12-2112 nucleotides that
encode the polypeptides of SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO:
42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ
ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO:
51, SEQ ID NO: 52, SEQ ID NO; 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ
ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO:
60, SEQ ID NO; 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ
ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO:
69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ
ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO:
78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ
ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO:
87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ
ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO:
96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100,
SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ
ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID
NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO:
113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO:
117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO:
121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO:
125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO:
129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO:
133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO:
137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO:
141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO:
145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO:
149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO:
153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO:
157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO:
161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO:
165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO:
169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO:
173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO:
177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO:
181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO:
185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO:
189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO:
193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO:
197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO:
201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO:
205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO:
209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO:
213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO:
217, SEQ ID NO: 218, SEQ ID NO: 219, or SEQ ID NO: 220, wherein the
nucleic acid includes the coding sequence for the mutation in the
NS3 protease domain of the above NS3/NS4A polypeptides, or a
complement thereof, (e.g., 12-15, 15-20, 20-30, 30-50, 50-100,
100-200, 200-500, 500-1000, 1000-1500, 1500-2079, or 1500-2112
consecutive nucleotides).
[0038] Methods of making and using the compositions described
herein are also provided. In addition to methods of making the
embodied nucleic acids and peptides, other embodiments include
methods of making immunogens and/or vaccine compositions that can
be used to treat or prevent HCV infection. Some methods are
practiced, for example, by mixing an adjuvant with a peptide or
nucleic acid antigen (e.g., an HCV peptide or HCV nucleic acid), as
described above, so as to formulate a single composition (e.g., a
vaccine composition). Preferred methods involve the mixing of
ribavirin with an HCV gene or antigen disclosed herein.
[0039] Preferred methods of using the compositions described herein
involve providing an animal in need of an immune response to HCV
with a sufficient amount of one or more of the nucleic acid or
peptide embodiments described herein. By one approach, for example,
an animal in need of an immune response to HCV (e.g., an animal at
risk or already infected with HCV, such as a human) is identified
and said animal is provided an amount of NS3/4A (SEQ. ID. NO.: 2 or
SEQ. ID. NO.: 36), a mutant NS3/4A (SEQ. ID. NOs.: 3-13), a
fragment thereof (e.g., SEQ. ID. NOs.: 14-26) or a nucleic acid
encoding said molecules that is effective to enhance or facilitate
an immune response to the hepatitis viral antigen. Additional
methods are practiced by identifying an animal in need of a potent
immune response to HCV and providing said animal a composition
comprising a peptide comprising an antigen or epitope present on
SEQ. ID. NOs.: 2-27 or SEQ. ID. NO.: 36 or a nucleic acid encoding
said peptides. Particularly preferred methods involve the
identification of an animal in need of an immune response to HCV
and providing said animal a composition comprising an amount of HCV
antigen (e.g., NS3/4A (SEQ. ID. NO.: 2 or SEQ. ID. NO.: 36)),
mutant NS3/4A (SEQ. ID. NOs.: 3-13), a fragment thereof containing
any number of consecutive amino acids between at least 3-50 amino
acids (e.g., 3, 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, or 50
consecutive amino acids) of SEQ. ID. NO.: 2 or SEQ. ID. NO.: 36
(e.g., SEQ. ID. NOs.: 14-26) or a nucleic acid encoding one or more
of these molecules that is sufficient to enhance or facilitate an
immune response to said antigen. In some embodiments, the
composition described above also contains an amount of ribavirin
that provides an adjuvant effect.
[0040] Other approaches concern identifying an animal in need of an
immune response to HCV and providing an amount of NS3/4A
polypeptides with altered protease activity, or mutations in the
NS3 protease domain (e.g., SEQ. ID. NO SEQ ID NO: 40, SEQ ID NO:
41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ
ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO:
50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO; 53, SEQ ID NO: 54, SEQ
ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO:
59, SEQ ID NO: 60, SEQ ID NO; 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ
ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO:
68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ
ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO:
77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ
ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO:
86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ
ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO:
95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ
ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID
NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO:
108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO:
112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO:
116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO:
120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO:
124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO:
128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO:
132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO:
136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO:
140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO:
144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO:
148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO:
152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO:
156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO:
160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO:
164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO:
168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO:
172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO:
176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO:
180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO:
184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO:
188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO:
192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO:
196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO:
200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO:
204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO:
208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO:
212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO:
216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, or SEQ ID NO:
220, a fragment thereof, or a nucleic acid encoding said molecules
that is effective to enhance or facilitate an immune response to
the hepatitis viral antigen. Additional methods are practiced by
identifying an animal in need of a potent immune response to HCV
and providing said animal a composition comprising a peptide
comprising an antigen or epitope present on SEQ. ID. NO SEQ ID NO:
40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ
ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO:
49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO; 53, SEQ
ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO:
58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO; 61, SEQ ID NO: 62, SEQ
ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO:
67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ
ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO:
76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ
ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO:
85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ
ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO:
94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ
ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID
NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO:
107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO:
111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO:
115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO:
119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO:
123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO:
127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO:
131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO:
135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO:
139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO:
143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO:
147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO:
151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO:
155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO:
159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO:
163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO:
167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO:
171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO:
175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO:
179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO:
183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO:
187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO:
191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO:
195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO:
199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO:
203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO:
207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO:
211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO:
215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO:
219, or SEQ ID NO: 220 or a nucleic acid encoding said peptides.
Particularly preferred methods involve the identification of an
animal in need of an immune response to HCV and providing said
animal a composition comprising an amount of HCV antigen (e.g. SEQ.
ID. NO SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43,
SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID
NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52,
SEQ ID NO; 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID
NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO; 61,
SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID
NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70,
SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID
NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79,
SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID
NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88,
SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID
NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97,
SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ
ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID
NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO:
110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO:
114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO:
118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO:
122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO:
126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO:
130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO:
134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO:
138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO:
142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO:
146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO:
150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO:
154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO:
158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO:
162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO:
166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO:
170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO:
174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO:
178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO:
182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO:
186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO:
190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO:
194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO:
198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO:
202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO:
206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO:
210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO:
214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO:
218, SEQ ID NO: 219, SEQ ID NO: 220), a fragment thereof containing
any number of consecutive amino acids between at least 3-50 amino
acids, wherein the fragment includes the mutation in the NS3
protease domain, (e.g., 3, 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40,
45, or 50 consecutive amino acids), or a nucleic acid encoding one
or more of these molecules that is sufficient to enhance or
facilitate an immune response to said antigen. In some embodiments,
the composition described above also contains an amount of
ribavirin that provides an adjuvant effect.
[0041] In still more embodiments, for example, a gene gun is used
to administer an HCV nucleic acid described herein (e.g., SEQ. ID.
NO.: 35 or fragment thereof, as described above) to a mammalian
subject in need of an immune response to HCV. In some embodiments,
an amount of ribavirin is mixed with the DNA immunogen prior to
delivery with the gene gun. In other embodiments, the DNA immunogen
is provided by gene gun shortly before or after administration of
ribavirin at or near the same site of DNA inoculation. For example,
in some embodiments, a gene gun or a transdermal delivery system is
used to administer HCV nucleic acids including nucleic acids
encoding the NS3/NS4A polypeptides with altered protease activity.
Accordingly, a gene gun or a transdermal delivery system is used to
deliver nucleotides encoding the NS3/NS4A polypeptides of SEQ ID
NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44,
SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID
NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO; 53,
SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID
NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO; 61, SEQ ID NO: 62,
SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID
NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71,
SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID
NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80,
SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID
NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89,
SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID
NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98,
SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ
ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID
NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO:
111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO:
115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO:
119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO:
123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO:
127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO:
131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO:
135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO:
139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO:
143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO:
147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO:
151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO:
155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO:
159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO:
163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO:
167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO:
171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO:
175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO:
179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO:
183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO:
187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO:
191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO:
195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO:
199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO:
203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO:
207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO:
211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO:
215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO:
219, and SEQ ID NO: 220, or fragments thereof that contain the
mutation in the NS3 protease domain, either with or without
ribavirin.
[0042] In still other embodiments, transdermal delivery systems are
used to deliver an HCV polypeptide described herein to a mammalian
subject in need of an immune response to HCV. In some embodiments,
an amount of ribavirin is mixed and delivered transdermally with
the HCV polypeptide. In other embodiments, an amount of ribavirin
is transdermally delivered to a mammal shortly before or shortly
after transdermal delivery of the HCV polypeptide, at or near the
same site as the polypeptide. For example, embodiments include
transdermal delivery of NS3/NS4A polypeptides having altered
protease activity. Thus, in some embodiments, the NS3/NS4A
polypeptides of SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID
NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47,
SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID
NO: 52, SEQ ID NO; 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56,
SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID
NO; 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65,
SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID
NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74,
SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID
NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83,
SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID
NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92,
SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID
NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO:
101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO:
105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO:
109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO:
113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO:
117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO:
121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO:
125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO:
129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO:
133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO:
137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO:
141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO:
145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO:
149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO:
153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO:
157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO:
161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO:
165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO:
169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO:
173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO:
177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO:
181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO:
185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO:
189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO:
193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO:
197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO:
201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO:
205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO:
209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO:
213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO:
217, SEQ ID NO: 218, SEQ ID NO: 219, and SEQ ID NO: 220 are
delivered transdermally to a mammalian subject in need of an immune
response to HCV, either with or without ribavirin.
[0043] Some embodiments relate to methods of inducing an immune
response to Hepatitis C Virus using any of the peptides, nucleic
acids, nucleic acids encoding said peptides, peptide fragments, or
nucleic acids encoding said peptide fragments described herein.
[0044] Other embodiments relate to methods of inducing an immune
response to heterologous antigen joined to or co-administered with
the peptides, nucleic acids, nucleic acids encoding said peptides,
peptide fragments, or nucleic acids encoding said peptide fragments
described herein. In some aspects the heterologous antigen is
chemically fused to the peptides, nucleic acids, nucleic acids
encoding said peptides, peptide fragments, or nucleic acids
encoding said peptide fragments described herein. In other aspects
the heterologous antigen is co-administered to the peptides,
nucleic acids, nucleic acids encoding said peptides, peptide
fragments, or nucleic acids encoding said peptide fragments
described herein. In still other aspects the heterologous antigen
is administered before administration of the peptides, nucleic
acids, nucleic acids encoding said peptides, peptide fragments, or
nucleic acids encoding said peptide fragments described herein. In
other aspects the heterologous antigen is administered after
administration of the peptides, nucleic acids, nucleic acids
encoding said peptides, peptide fragments, or nucleic acids
encoding said peptide fragments described herein.
[0045] In some aspects, nucleic acids encoding heterologous
antigens described herein that are joined to or co-administered
with the nucleic acids, nucleic acids encoding said peptides,
peptide fragments, or nucleic acids encoding said peptide fragments
described herein are at least, at least about, less than, or less
than about 3 nucleotides, 4 nucleotides, 5 nucleotides, 6
nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10
nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14
nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18
nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22
nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26
nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30
nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34
nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38
nucleotides, 39 nucleotides, 40 nucleotides, 41 nucleotides, 42
nucleotides, 43 nucleotides, 44 nucleotides, 45 nucleotides, 46
nucleotides, 47 nucleotides, 48 nucleotides, 49 nucleotides, 50
nucleotides, 55 nucleotides, 60 nucleotides, 65 nucleotides, 70
nucleotides, 75 nucleotides, 80 nucleotides, 85 nucleotides, 90
nucleotides, 95 nucleotides, 100 nucleotides, 110 nucleotides, 120
nucleotides, 130 nucleotides, 140 nucleotides, 150 nucleotides, 160
nucleotides, 170 nucleotides, 180 nucleotides, 190 nucleotides, 200
nucleotides, 250 nucleotides, 300 nucleotides, 350 nucleotides, 400
nucleotides, 450 nucleotides, 500 nucleotides, 550 nucleotides, 600
nucleotides, 650 nucleotides, 700 nucleotides, 750 nucleotides, 800
nucleotides, 850 nucleotides, 900 nucleotides, 950 nucleotides,
1000 nucleotides, 1100 nucleotides, 1200 nucleotides, 1300
nucleotides, 1400 nucleotides, 1500 nucleotides, 1600 nucleotides,
1700 nucleotides, 1800 nucleotides, 1900 nucleotides, 2000
nucleotides, 2500 nucleotides, 3000 nucleotides, 3500 nucleotides,
4000 nucleotides, 4500 nucleotides, 5000 nucleotides, 6000
nucleotides, 7000 nucleotides, 8000 nucleotides, 9000 nucleotides,
10,000 nucleotides in length.
[0046] In some aspects, heterologous antigens described herein that
are joined to or co-administered with the nucleic acids, nucleic
acids encoding said peptides, peptide fragments, or nucleic acids
encoding said peptide fragments described herein are at least, at
least about, less than, or less than about 3 amino acids, 4 amino
acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids,
9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13
amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17
amino acids, 18 amino acids, 19 amino acids, 20 amino acids, 21
amino acids, 22 amino acids, 23 amino acids, 24 amino acids, 25
amino acids, 26 amino acids, 27 amino acids, 28 amino acids, 29
amino acids, 30 amino acids, 31 amino acids, 32 amino acids, 33
amino acids, 34 amino acids, 35 amino acids, 36 amino acids, 37
amino acids, 38 amino acids, 39 amino acids, 40 amino acids, 41
amino acids, 42 amino acids, 43 amino acids, 44 amino acids, 45
amino acids, 46 amino acids, 47 amino acids, 48 amino acids, 49
amino acids, 50 amino acids, 55 amino acids, 60 amino acids, 65
amino acids, 70 amino acids, 75 amino acids, 80 amino acids, 85
amino acids, 90 amino acids, 95 amino acids, 100 amino acids, 110
amino acids, 120 amino acids, 130 amino acids, 140 amino acids, 150
amino acids, 160 amino acids, 170 amino acids, 180 amino acids, 190
amino acids, 200 amino acids, 250 amino acids, 300 amino acids, 350
amino acids, 400 amino acids, 450 amino acids, 500 amino acids, 550
amino acids, 600 amino acids, 650 amino acids, 700 amino acids, 750
amino acids, 800 amino acids, 850 amino acids, 900 amino acids, 950
amino acids, 1000 amino acids, 1100 amino acids, 1200 amino acids,
1300 amino acids, 1400 amino acids, 1500 amino acids, 1600 amino
acids, 1700 amino acids, 1800 amino acids, 1900 amino acids, 2000
amino acids, 2500 amino acids, 3000 amino acids, 3500 amino acids,
4000 amino acids, 4500 amino acids, 5000 amino acids, 6000 amino
acids, 7000 amino acids, 8000 amino acids, 9000 amino acids, 10,000
amino acids in length.
[0047] Still other embodiments relate to methods of inducing an
immune response as described herein when cleavage of IPS-1 to
.DELTA.IPS-1 is not desired.
[0048] Other embodiments relate to methods of inducing an immune
response as described herein wherein repression of IFN.alpha.
and/or IFN.beta. is not desired. This is particularly useful when
raising an immune response to HCV because HCV NS3/4A represses
IFN.alpha. and IFN.beta. expression through protetolytic cleavage
of IPS-1.
[0049] One embodiment relates to a method of enhancing an immune
response to a hepatitis C antigen comprising identifying an animal
in need of an enhanced immune response to a hepatitis C antigen and
providing to said animal a composition comprising a nucleic acid
sequence that encodes a peptide comprising an NS3 protease domain
that cleaves NS3-NS4A cleavage site but does not cleave IPS-1 to
.DELTA.IPS-1.
[0050] Another embodiment relates to a method of enhancing an
immune response to a hepatitis C antigen comprising identifying an
animal in need of an enhanced immune response to a hepatitis C
antigen and providing to said animal a peptide that comprises an
NS3 protease domain that cleaves NS3-NS4A cleavage site but does
not cleave IPS-1 to .DELTA.IPS-1. In one aspect of the embodiments,
the composition further comprises ribavirin. In another aspect of
the embodiments, the peptide used is selected from the group
consisting of SEQ ID NO: 68, SEQ ID NO: 73, SEQ ID NO: 1329, SEQ ID
NO: 1330, SEQ ID NO: 1331, SEQ ID NO: 1332, SEQ ID NO: 1333, SEQ ID
NO: 1334, SEQ ID NO: 1335, SEQ ID NO: 1336, SEQ ID NO: 1337, SEQ ID
NO: 1338, and SEQ ID NO: 1339.
[0051] Another embodiment relates to a method of enhancing an
immune response to a hepatitis C antigen comprising identifying an
animal in need of an enhanced immune response to a hepatitis C
antigen and providing to said animal a composition comprising at
least 100 consecutive nucleotides of a nucleic acid sequence that
encodes a peptide comprising an NS3 protease domain that cleaves
NS3-NS4A cleavage site but does not cleave IPS-1 to .DELTA.IPS-1,
wherein said nucleic acid codes for a peptide fragment that retains
the ability to cleave the NS3-NS4A cleavage site but not cleave
IPS-1 to .DELTA.IPS-1.
[0052] Still another embodiment relates to a method of enhancing an
immune response to a hepatitis C antigen comprising identifying an
animal in need of an enhanced immune response to a hepatitis C
antigen and providing to said animal a composition comprising a
peptide comprising at least 34 amino acids of an NS3 protease
domain that cleaves NS3-NS4A cleavage site but does not cleave
IPS-1 to .DELTA.IPS-1, wherein said peptide fragment retains the
ability to cleave the NS3-NS4A cleavage site but not cleave IPS-1
to .DELTA.IPS-1.
[0053] One embodiment relates to a purified or isolated nucleic
acid encoding a polypeptide comprising an NS3/NS4A polypeptide that
cleaves NS3-NS4A cleavage site but cannot cleave IPS-1 to
.DELTA.IPS-1 wherein the polypeptide is SEQ ID NO. 1329.
[0054] In one aspect, the mutant NS3/4A peptide has altered
cleavage specificity for host proteins. In another aspect, the
nucleic acid is part of a composition. In some aspects, the
composition further comprises ribavirin.
[0055] One embodiment relates to a purified or isolated nucleic
acid encoding a mutant NS/4A peptide with retained protease
activity, wherein said mutant NS3/4A peptide has altered cleavage
specificity for host proteins.
[0056] One embodiment relates to a method of enhancing an immune
response to a hepatitis C antigen comprising identifying an animal
in need of an enhanced immune response to a hepatitis C antigen;
and administering to said animal a composition a purified or
isolated nucleic acid encoding a mutant NS/4A peptide with retained
protease activity, wherein said mutant NS3/4A peptide has altered
cleavage specificity for host proteins. In one aspect, the
composition further comprises ribavirin. In another aspect of the
method, ribavirin is co-administered. In yet another aspect, the
co-administration of ribavirin is immediately before or immediately
after the administration of said composition. In yet another aspect
of the embodiments described herein, the site of said
administration, or a region proximal to said site of
administration, is contacted with an electrode configured for
electroporation of a nucleic acid.
[0057] Vectors that include the isolated nucleic acids or that
encode the isolated polypeptides described herein are also
embodiments. Compositions that include the nucleic acids,
polypeptides, and vectors described herein (e.g., vials, tubes,
hypodermic needles, electroporation devices, oils, and transdermal
delivery compositions) are also embodiments. Cells that include
these vectors are also provided herein. For example, in some
embodiments, the nucleic acids described herein are inserted into
an expression vector such as a pVAX.TM. expression vector or are
inserted into a Semliki forest virus vector and these expression
constructs are provided to a subject in need of an immune response
to the antigen.
[0058] Stated differently, several embodiments described herein
concern nucleic acids that encode a recombinant HCV NS3/4A chimeric
molecule, wherein the nucleic acid encodes four domains (domain W,
domain X, domain Y, and domain Z). Domain W can encode an NS3
protease domain prepared as described herein (e.g., a mutant or
modified version), or fragment thereof, domain X can encode an NS3
helicase domain, or a fragment thereof, and domain Y can encode an
NS4A co-factor domain, or a fragment thereof. Domain Z can encode
an antigen, an epitope of a pathogen, a TCE, and/or the
aforementioned molecules with or without a linker and/or adjuvant
sequence. In preferred embodiments, the recombinant HCV NS3/4A
chimeric polypeptide retains catalytic activity (e.g., protease,
helicase, or protease and helicase activity). In preferred
embodiments, domain Z is in a position within the recombinant
nucleic acid and encoded chimeric polypeptide, which is
non-naturally-occurring (e.g., when Z encodes an HCV antigen).
[0059] In some embodiments, the W domain comprises the nucleic acid
sequence of residues 1-551 of an NS3/4A sequence such as SEQ ID
NOs: 1 or 35, or an analogous sequence of any NS3/4A nucleic acid,
or a fragment thereof (e.g., said fragment can comprise, consist
of, or consist essentially of about at least, equal to, greater
than, less than, or any number in between 9, 15, 30, 50, 75, 100,
125, 150, 175, 200, 250, or 300 consecutive nucleotides of the
protease domain of SEQ. ID. Nos. 1 or 35). In some embodiments, the
X domain can comprise the nucleic acid sequence of residues
218-1568 of SEQ ID NOs: 1 or 35, or an analogous sequence of any
NS3/4A nucleic acid, or a fragment thereof (e.g., said fragment can
comprise, consist of, or consist essentially of about at least,
equal to, greater than, less than, or any number in between 9, 15,
30, 50, 75, 100, 125, 150, 175, 200, 250, or 300 consecutive
nucleotides of the helicase domain of SEQ. ID. Nos. 1 or 35). In
some embodiments, the Y domain can comprise the nucleic acid
sequence of residues 1569-2069 of SEQ ID NOs: 1 or 35, or an
analogous sequence of any NS3/4A nucleic acid, or a fragment
thereof (e.g., said fragment can comprise, consist of, or consist
essentially of about at least, equal to, greater than, less than,
or any number in between 9, 15, 30, 50, 75, 100, 125, 150, 175,
200, 250, 300, or 350 consecutive nucleotides of the NS4A domain of
SEQ. ID. Nos. 1 or 35). In preferred embodiments, the Z domain can
comprise the nucleic acid sequence of an antigen, preferably a TCE,
and more preferably a sequence selected from the group consisting
of SEQ ID NOs: 221-571 and SEQ ID NOs: 809-1011 and SEQ ID NO:
1014.
[0060] In some embodiments, the Z domain is located within or
flanking (e.g., juxtaposed or immediately adjacent to) said W
domain. For example, the nucleic acid encoding the Z domain can
place the encoded antigen between or next to amino acids 1 and 2, 2
and 3, 3 and 4, 4 and 5, 5 and 6, 6 and 7, 7 and 8, 8 and 9, 9 and
10, 10 and 11, 11 and 12, 12 and 13, 13 and 14, 14 and 15, 15 and
16, 16 and 17, 17 and 18, 18 and 19, 19 and 20, 20 and 21, 21 and
22, 22 and 23, 23 and 24, 24 and 25, 25 and 26, 26 and 27, 27 and
28, 28 and 29, 29 and 30, 30 and 31, 31 and 32, 32 and 33, 33 and
34, 34 and 35, 35 and 36, 36 and 37, 37 and 38, 38 and 39, 39 and
40, 40 and 41, 41 and 42, 42 and 43, 43 and 44, 44 and 45, 45 and
46, 46 and 47, 47 and 48, 48 and 49, 49 and 50, 50 and 51, 51 and
52, 52 and 53, 53 and 54, 54 and 55, 55 and 56, 56 and 57, 57 and
58, 58 and 59, 59 and 60, 60 and 61, 61 and 62, 62 and 63, 63 and
64, 64 and 65, 65 and 66, 66 and 67, 67 and 68, 68 and 69, 69 and
70, 70 and 71, 71 and 72, 72 and 73, 73 and 74, 74 and 75, 75 and
76, 76 and 77, 77 and 78, 78 and 79, 79 and 80, 80 and 81, 81 and
82, 82 and 83, 83 and 84, 84 and 85, 85 and 86, 86 and 87, 87 and
88, 88 and 89, 89 and 90, 90 and 91, 91 and 92, 92 and 93, 93 and
94, 94 and 95, 95 and 96, 96 and 97, 97 and 98, 98 and 99, 99 and
100, 100 and 101, 101 and 102, 102 and 103, 103 and 104, 104 and
105, 105 and 106, 106 and 107, 107 and 108, 108 and 109, 109 and
110, 110 and 111, 111 and 112, 112 and 113, 113 and 114, 114 and
115, 115 and 116, 116 and 117, 117 and 118, 118 and 119, 119 and
120, 120 and 121, 121 and 122, 122 and 123, 123 and 124, 124 and
125, 125 and 126, 126 and 127, 127 and 128, 128 and 129, 129 and
130, 130 and 131, 131 and 132, 132 and 133, 133 and 134, 134 and
135, 135 and 136, 136 and 137, 137 and 138, 138 and 139, 139 and
140, 140 and 141, 141 and 142, 142 and 143, 143 and 144, 144 and
145, 145 and 146, 146 and 147, 147 and 148, 148 and 149, 149 and
150, 150 and 151, 151 and 152, 152 and 153, 153 and 154, 154 and
155, 155 and 156, 156 and 157, 157 and 158, 158 and 159, 159 and
160, 160 and 161, 161 and 162, 162 and 163, 163 and 164, 164 and
165, 165 and 166, 166 and 167, 167 and 168, 168 and 169, 169 and
170, 170 and 171, 171 and 172, 172 and 173, 173 and 174, 174 and
175, 175 and 176, 176 and 177, 177 and 178, 178 and 179, 179 and
180, or 180 and 181 of said W domain of said chimeric
polypeptide.
[0061] In some embodiments, the Z domain is located within or
flanking (e.g., juxtaposed or immediately adjacent to) the X
domain. For example, the Z domain can place the encoded antigen
between or next to amino acids 1 and 2, 2 and 3, 3 and 4, 4 and 5,
5 and 6, 6 and 7, 7 and 8, 8 and 9, 9 and 10, 10 and 11, 11 and 12,
12 and 13, 13 and 14, 14 and 15, 15 and 16, 16 and 17, 17 and 18,
18 and 19, 19 and 20, 20 and 21, 21 and 22, 22 and 23, 23 and 24,
24 and 25, 25 and 26, 26 and 27, 27 and 28, 28 and 29, 29 and 30,
30 and 31, 31 and 32, 32 and 33, 33 and 34, 34 and 35, 35 and 36,
36 and 37, 37 and 38, 38 and 39, 39 and 40, 40 and 41, 41 and 42,
42 and 43, 43 and 44, 44 and 45, 45 and 46, 46 and 47, 47 and 48,
48 and 49, 49 and 50, 50 and 51, 51 and 52, 52 and 53, 53 and 54,
54 and 55, 55 and 56, 56 and 57, 57 and 58, 58 and 59, 59 and 60,
60 and 61, 61 and 62, 62 and 63, 63 and 64, 64 and 65, 65 and 66,
66 and 67, 67 and 68, 68 and 69, 69 and 70, 70 and 71, 71 and 72,
72 and 73, 73 and 74, 74 and 75, 75 and 76, 76 and 77, 77 and 78,
78 and 79, 79 and 80, 80 and 81, 81 and 82, 82 and 83, 83 and 84,
84 and 85, 85 and 86, 86 and 87, 87 and 88, 88 and 89, 89 and 90,
90 and 91, 91 and 92, 92 and 93, 93 and 94, 94 and 95, 95 and 96,
96 and 97, 97 and 98, 98 and 99, 99 and 100, 100 and 101, 101 and
102, 102 and 103, 103 and 104, 104 and 105, 105 and 106, 106 and
107, 107 and 108, 108 and 109, 109 and 110, 110 and 111, 111 and
112, 112 and 113, 113 and 114, 114 and 115, 115 and 116, 116 and
117, 117 and 118, 118 and 119, 119 and 120, 120 and 121, 121 and
122, 122 and 123, 123 and 124, 124 and 125, 125 and 126, 126 and
127, 127 and 128, 128 and 129, 129 and 130, 130 and 131, 131 and
132, 132 and 133, 133 and 134, 134 and 135, 135 and 136, 136 and
137, 137 and 138, 138 and 139, 139 and 140, 140 and 141, 141 and
142, 142 and 143, 143 and 144, 144 and 145, 145 and 146, 146 and
147, 147 and 148, 148 and 149, 149 and 150, 150 and 151, 151 and
152, 152 and 153, 153 and 154, 154 and 155, 155 and 156, 156 and
157, 157 and 158, 158 and 159, 159 and 160, 160 and 161, 161 and
162, 162 and 163, 163 and 164, 164 and 165, 165 and 166, 166 and
167, 167 and 168, 168 and 169, 169 and 170, 170 and 171, 171 and
172, 172 and 173, 173 and 174, 174 and 175, 175 and 176, 176 and
177, 177 and 178, 178 and 179, 179 and 180, 180 and 181, 181 and
182, 182 and 183, 183 and 184, 184 and 185, 185 and 186, 186 and
187, 187 and 188, 188 and 189, 189 and 190, 190 and 191, 191 and
192, 192 and 193, 193 and 194, 194 and 195, 195 and 196, 196 and
197, 197 and 198, 198 and 199, 199 and 200, 200 and 201, 201 and
202, 202 and 203, 203 and 204, 204 and 205, 205 and 206, 206 and
207, 207 and 208, 208 and 209, 209 and 210, 210 and 211, 211 and
212, 212 and 213, 213 and 214, 214 and 215, 215 and 216, 216 and
217, 217 and 218, 218 and 219, 219 and 220, 220 and 221, 221 and
222, 222 and 223, 223 and 224, 224 and 225, 225, and 226, 226 and
227, 227 and 228, 228 and 229, 229 and 230, 230 and 231, 231 and
232, 232 and 233, 233 and 234, 234 and 235, 235 and 236, 236 and
237, 237 and 238, 238 and 239, 239 and 240, 240 and 241, 241 and
242, 242 and 243, 243 and 244, 244 and 245, 245 and 246, 246 and
247, 247 and 248, 248 and 249, 249 and 250, 250 and 251, 251 and
252, 252 and 253, 253 and 254, 254 and 255, 255 and 256, 256 and
257, 257 and 258, 258 and 259, 259 and 260, 260 and 261, 261 and
262, 262 and 263, 263 and 264, 264 and 265, 265 and 266, 266 and
267, 267 and 268, 268 and 269, 269 and 270, 270 and 271, 271 and
272, 272 and 273, 273 and 274, 274 and 275, 275 and 276, 276 and
277, 277 and 278, 278 and 279, 279 and 280, 280 and 281, 281 and
282, 282 and 283, 283 and 284, 284 and 285, 285 and 286, 286 and
287, 287 and 288, 288 and 289, 289 and 290, 290 and 291, 291 and
292, 292 and 293, 293 and 294, 294 and 295, 295 and 296, 296 and
297, 297 and 298, 298 and 299, 299 and 300, 300 and 201, 301 and
302, 302 and 303, 303 and 304, 304 and 305, 305 and 306, 306 and
307, 307 and 308, 308 and 309, 309 and 310, 310 and 311, 311 and
312, 312 and 313, 313 and 314, 314 and 315, 315 and 316, 316 and
317, 317 and 318, 318 and 319, 319 and 320, 320 and 321, 321, and
322, 322 and 323, 323 and 324, 324 and 325, 325, and 326, 326 and
327, 327 and 328, 328 and 329, 329 and 330, 330 and 331, 331 and
332, 332 and 333, 333 and 334, 334 and 335, 335 and 336, 336 and
337, 337 and 338, 338 and 339, 339 and 340, 340 and 341, 341 and
342, 342 and 343, 343 and 344, 344 and 345, 345 and 346, 346 and
347, 347 and 348, 348 and 349, 349 and 350, 350 and 351, 351 and
352, 352 and 353, 353 and 354, 354 and 355, 355 and 356, 356 and
357, 357 and 358, 358 and 359, 359 and 360, 360 and 361, 361 and
362, 362 and 363, 363 and 364, 364 and 365, 365 and 366, 366 and
367, 367 and 368, 368 and 369, 369 and 370, 370 and 371, 371 and
372, 372 and 373, 373 and 374, 374 and 375, 375 and 376, 376 and
377, 377 and 378, 378 and 379, 379 and 380, 380 and 381, 381 and
382, 382 and 383, 383 and 384, 384 and 385, 385 and 386, 386 and
387, 387 and 388, 388 and 389, 389 and 390, 390 and 391, 391 and
392, 392 and 393, 393 and 394, 394 and 395, 395 and 396, 396 and
397, 397 and 398, 398 and 399, 399 and 400, 401 and 402, 402 and
403, 403 and 404, 404 and 405, 405 and 406, 406 and 407, 407 and
408, 408 and 409, 409 and 410, 410 and 411, 411 and 412, 412 and
413, 413 and 414, 414 and 415, 415 and 416, 416 and 417, 417 and
418, 418 and 419, 419 and 420, 420 and 421, 421 and 422, 422 and
423, 423 and 424, 424 and 425, 425 and 426, 426 and 427, 427 and
428, 428 and 429, 429 and 430, 430 and 431, 431 and 432, 432 and
433, 433 and 434, 434 and 435, 435 and 436, 436 and 437, 437 and
438, 438 and 439, 439 and 440, 440 and 441, 441 and 442, 442 and
443, 443 and 444, 444 and 445, 445 and 446, 446 and 447, 447 and
448, 448 and 449, or 449 and 450 of the X domain of the chimeric
polypeptide. Preferably, the Z domain is located between amino
acids 383 and 450 of said X domain.
[0062] Optionally, the Z domain is located within or flanking
(e.g., juxtaposed or immediately adjacent to) said Y domain. For
example, the Z domain can place the encoded antigen between or next
to amino acids 1 and 2, 2 and 3, 3 and 4, 4 and 5, 5 and 6, 6 and
7, 7 and 8, 8 and 9, 9 and 10, 10 and 11, 11 and 12, 12 and 13, 13
and 14, 14 and 15, 15 and 16, 16 and 17, 17 and 18, 18 and 19, 19
and 20, 20 and 21, 21 and 22, 22 and 23, 23 and 24, 24 and 25, 25
and 26, 26 and 27, 27 and 28, 28 and 29, 29 and 30, 30 and 31, 31
and 32, 32 and 33, 33 and 34, 34 and 35, 35 and 36, 36 and 37, 37
and 38, 38 and 39, 39 and 40, 40 and 41, 41 and 42, 42 and 43, 43
and 44, 44 and 45, 45 and 46, 46 and 47, 47 and 48, 48 and 49, 49
and 50, 50 and 51, 51 and 52, 52 and 53, or 53 and 54 of the Y
domain of the chimeric polypeptide.
[0063] That is, aspects of the invention concern a composition that
comprises, consists of, or consists essentially of an isolated
nucleic acid provided by the formula:
WXYZ
[0064] wherein:
[0065] W encodes a protease domain (e.g., an HCV NS3 protease
domain prepared as described herein, such as a mutant with enhanced
or altered protease activity or substrate specificity) or a
fragment thereof, wherein said fragment comprises, consists of, or
consists essentially of about at least, equal to, greater than,
less than, or any number in between 9, 15, 30, 50, 75, 100, 125,
150, 175, 200, 250, or 300 consecutive nucleotides of the protease
domain of SEQ. ID. Nos. 1 or 35 (e.g., residues 1-551 of SEQ ID NO:
35, or an analogous position in any NS3/4A nucleic acid);
[0066] X encodes a helicase domain (e.g., an HCV helicase domain)
or a fragment thereof, wherein said fragment comprises, consists
of, or consists essentially of about at least, equal to, greater
than, less than, or any number in between 9, 15, 30, 50, 75, 100,
125, 150, 175, 200, 250, or 300 consecutive nucleotides of the
helicase domain of SEQ. ID. Nos. 1 or 35 (e.g., residues 218-1568
of SEQ ID NO: 35, or an analogous position in any NS3/4A nucleic
acid);
[0067] Y encodes an enhancer domain (e.g., an HCV NS4A domain) or a
fragment thereof, wherein said fragment comprises, consists of, or
consists essentially of about at least, equal to, greater than,
less than, or any number in between 9, 15, 30, 50, 75, 100, 125,
150, 175, 200, 250, or 300 consecutive nucleotides of the NS4A
domain of SEQ. ID. Nos. 1 or 35 (e.g., residues 1569-2069 of SEQ ID
NO: 35, or an analogous position in any NS3/4A nucleic acid);
and
[0068] Z encodes an antigen (e.g., an antigen of a virus, bacteria,
toxin, or cancer cell, such as a TCE provided by a sequence
selected from the group consisting of SEQ ID NOs: 221-571, SEQ ID
NOs: 809-1011, SEQ ID NO: 1014, SEQ ID NOs: 1016-1034, SEQ ID NOs:
1146-1173, and SEQ ID NOs: 1210-1328), [0069] with the proviso that
Z is not in a position that is naturally occurring in HCV (e.g.,
when Z is an HCV antigen, the antigen is inserted at a position
that is not naturally occurring in HCV).
[0070] Aspects of the invention also concern a composition that
comprises, consists of, or consists essentially of an isolated
nucleic acid provided by the formula:
ZWXY
[0071] wherein:
[0072] W encodes a protease domain (e.g., an HCV NS3 protease
domain prepared as described herein, such as a mutant with enhanced
or altered protease activity or substrate specificity) or a
fragment thereof, wherein said fragment comprises, consists of, or
consists essentially of about at least, equal to, greater than,
less than, or any number in between 9, 15, 30, 50, 75, 100, 125,
150, 175, 200, 250, or 300 consecutive nucleotides of the protease
domain of SEQ. ID. Nos. 1 or 35 (e.g., residues 1-551 of SEQ ID NO:
35, or an analogous position in any NS3/4A nucleic acid);
[0073] X encodes a helicase domain (e.g., an HCV helicase domain)
or a fragment thereof, wherein said fragment comprises, consists
of, or consists essentially of about at least, equal to, greater
than, less than, or any number in between 9, 15, 30, 50, 75, 100,
125, 150, 175, 200, 250, or 300 consecutive nucleotides of the
helicase domain of SEQ. ID. Nos. 1 or 35 (e.g., residues 218-1568
of SEQ ID NO: 35, or an analogous position in any NS3/4A nucleic
acid);
[0074] Y encodes an enhancer domain (e.g., an HCV NS4A domain) or a
fragment thereof, wherein said fragment comprises, consists of, or
consists essentially of about at least, equal to, greater than,
less than, or any number in between 9, 15, 30, 50, 75, 100, 125,
150, 175, 200, 250, or 300 consecutive nucleotides of the NS4A
domain of SEQ. ID. Nos. 1 or 35 (e.g., residues 1569-2069 of SEQ ID
NO: 35, or an analogous position in any NS3/4A nucleic acid);
and
[0075] Z encodes an antigen (e.g., an antigen of a virus, bacteria,
toxin, or cancer cell, such as a TCE provided by a sequence
selected from the group consisting of SEQ ID NOs: 221-571, SEQ ID
NOs: 809-1011, SEQ ID NO: 1014, SEQ ID NOs: 1016-1034, SEQ ID NOs:
1146-1173, and SEQ ID NOs: 1210-1328), [0076] with the proviso that
Z is not in a position that is naturally occurring in HCV (e.g.,
when Z is an HCV antigen, the antigen is inserted at a position
that is not naturally occurring in HCV).
[0077] Aspects of the invention also concern a composition that
comprises, consists of, or consists essentially of an isolated
nucleic acid provided by the formula:
WZXY
[0078] wherein:
[0079] W encodes a protease domain (e.g., an HCV NS3 protease
domain prepared as described herein, such as a mutant with enhanced
or altered protease activity or substrate specificity) or a
fragment thereof, wherein said fragment comprises, consists of, or
consists essentially of about at least, equal to, greater than,
less than, or any number in between 9, 15, 30, 50, 75, 100, 125,
150, 175, 200, 250, or 300 consecutive nucleotides of the protease
domain of SEQ. ID. Nos. 1 or 35 (e.g., residues 1-551 of SEQ ID NO:
35, or an analogous position in any NS3/4A nucleic acid);
[0080] X encodes a helicase domain (e.g., an HCV helicase domain)
or a fragment thereof, wherein said fragment comprises, consists
of, or consists essentially of about at least, equal to, greater
than, less than, or any number in between 9, 15, 30, 50, 75, 100,
125, 150, 175, 200, 250, or 300 consecutive nucleotides of the
helicase domain of SEQ. ID. Nos. 1 or 35 (e.g., residues 218-1568
of SEQ ID NO: 35, or an analogous position in any NS3/4A nucleic
acid);
[0081] Y encodes an enhancer domain (e.g., an HCV NS4A domain) or a
fragment thereof, wherein said fragment comprises, consists of, or
consists essentially of about at least, equal to, greater than,
less than, or any number in between 9, 15, 30, 50, 75, 100, 125,
150, 175, 200, 250, or 300 consecutive nucleotides of the NS4A
domain of SEQ. ID. Nos. 1 or 35 (e.g., residues 1569-2069 of SEQ ID
NO: 35, or an analogous position in any NS3/4A nucleic acid);
and
[0082] Z encodes an antigen (e.g., an antigen of a virus, bacteria,
toxin, or cancer cell, such as a TCE provided by a sequence
selected from the group consisting of SEQ ID NOs: 221-571, SEQ ID
NOs: 809-1011, SEQ ID NO: 1014, SEQ ID NOs: 1016-1034, SEQ ID NOs:
1146-1173, and SEQ ID NOs: 1210-1328), [0083] with the proviso that
Z is not in a position that is naturally occurring in HCV (e.g.,
when Z is an HCV antigen, the antigen is inserted at a position
that is not naturally occurring in HCV).
[0084] Aspects of the invention also concern a composition that
comprises, consists of, or consists essentially of an isolated
nucleic acid provided by the formula:
WZXY
[0085] wherein:
[0086] W encodes a protease domain (e.g., an HCV NS3 protease
domain prepared as described herein, such as a mutant with enhanced
or altered protease activity or substrate specificity) or a
fragment thereof, wherein said fragment comprises, consists of, or
consists essentially of about at least, equal to, greater than,
less than, or any number in between 9, 15, 30, 50, 75, 100, 125,
150, 175, 200, 250, or 300 consecutive nucleotides of the protease
domain of SEQ. ID. Nos. 1 or 35 (e.g., residues 1-551 of SEQ ID NO:
35, or an analogous position in any NS3/4A nucleic acid);
[0087] X encodes a helicase domain (e.g., an HCV helicase domain)
or a fragment thereof, wherein said fragment comprises, consists
of, or consists essentially of about at least, equal to, greater
than, less than, or any number in between 9, 15, 30, 50, 75, 100,
125, 150, 175, 200, 250, or 300 consecutive nucleotides of the
helicase domain of SEQ. ID. Nos. 1 or 35 (e.g., residues 218-1568
of SEQ ID NO: 35, or an analogous position in any NS3/4A nucleic
acid);
[0088] Y encodes an enhancer domain (e.g., an HCV NS4A domain) or a
fragment thereof, wherein said fragment comprises, consists of, or
consists essentially of about at least, equal to, greater than,
less than, or any number in between 9, 15, 30, 50, 75, 100, 125,
150, 175, 200, 250, or 300 consecutive nucleotides of the NS4A
domain of SEQ. ID. Nos. 1 or 35 (e.g., residues 1569-2069 of SEQ ID
NO: 35, or an analogous position in any NS3/4A nucleic acid);
and
[0089] Z encodes an antigen (e.g., an antigen of a virus, bacteria,
toxin, or cancer cell, such as a TCE provided by a sequence
selected from the group consisting of SEQ ID NOs: 221-571, SEQ ID
NOs: 809-1011, SEQ ID NO: 1014, SEQ ID NOs: 1016-1034, SEQ ID NOs:
1146-1173, and SEQ ID NOs: 1210-1328) [0090] with the proviso that
Z is not in a position that is naturally occurring in HCV (e.g.,
when Z is an HCV antigen, the antigen is inserted at a position
that is not naturally occurring in HCV).
[0091] Aspects of the invention also concern a composition that
comprises, consists of, or consists essentially of an isolated
nucleic acid provided by the formula:
WXZY
[0092] wherein:
[0093] W encodes a protease domain (e.g., an HCV NS3 protease
domain prepared as described herein, such as a mutant with enhanced
or altered protease activity or substrate specificity) or a
fragment thereof, wherein said fragment comprises, consists of, or
consists essentially of about at least, equal to, greater than,
less than, or any number in between 9, 15, 30, 50, 75, 100, 125,
150, 175, 200, 250, or 300 consecutive nucleotides of the protease
domain of SEQ. ID. Nos. 1 or 35 (e.g., residues 1-551 of SEQ ID NO:
35, or an analogous position in any NS3/4A nucleic acid);
[0094] X encodes a helicase domain (e.g., an HCV helicase domain)
or a fragment thereof, wherein said fragment comprises, consists
of, or consists essentially of about at least, equal to, greater
than, less than, or any number in between 9, 15, 30, 50, 75, 100,
125, 150, 175, 200, 250, or 300 consecutive nucleotides of the
helicase domain of SEQ. ID. Nos. 1 or 35 (e.g., residues 218-1568
of SEQ ID NO: 35, or an analogous position in any NS3/4A nucleic
acid);
[0095] Y encodes an enhancer domain (e.g., an HCV NS4A domain) or a
fragment thereof, wherein said fragment comprises, consists of, or
consists essentially of about at least, equal to, greater than,
less than, or any number in between 9, 15, 30, 50, 75, 100, 125,
150, 175, 200, 250, or 300 consecutive nucleotides of the NS4A
domain of SEQ. ID. Nos. 1 or 35 (e.g., residues 1569-2069 of SEQ ID
NO: 35, or an analogous position in any NS3/4A nucleic acid)
and
[0096] Z encodes an antigen (e.g., an antigen of a virus, bacteria,
toxin, or cancer cell, such as a TCE provided by a sequence
selected from the group consisting of SEQ ID NOs: 221-571, SEQ ID
NOs: 809-1011, SEQ ID NO: 1014, SEQ ID NOs: 1016-1034, SEQ ID NOs:
1146-1173, and SEQ ID NOs: 1210-1328), [0097] with the proviso that
Z is not in a position that is naturally occurring in HCV (e.g.,
when Z is an HCV antigen, the antigen is inserted at a position
that is not naturally occurring in HCV).
[0098] Several other embodiments described herein concern
recombinant HCV NS3/4A polypeptides, wherein the polypeptide
comprises four domains (domain W, domain X, domain Y, and domain
Z). Domain W can be an NS3 protease domain as described herein, or
fragment thereof, domain X can be an NS3 helicase domain, or a
fragment thereof, and domain Y can be an NS4A co-factor domain, or
a fragment thereof. Domain Z can be an antigen, an epitope of a
pathogen, a TCE, and/or the aforementioned molecules with or
without a linker and/or adjuvant sequence. In preferred
embodiments, the recombinant HCV NS3/4A chimeric polypeptide
retains catalytic activity (e.g., protease, helicase, or protease
and helicase activity) but may have altered activity or
specificity. In preferred embodiments, domain Z is in a position
within the recombinant chimeric polypeptide, which is
non-naturally-occurring (e.g., when Z encodes an HCV antigen).
[0099] In some embodiments, the W domain comprises the polypeptide
encoded nucleic acid sequence of residues 1-551 of an NS3/4A
sequence such as SEQ ID NOs: 1 or 35, or an analogous sequence
encoded by any NS3/4A nucleic acid, or a fragment thereof (e.g.,
said fragment can comprise, consist of, or consist essentially of a
polypeptide encoded by about at least, equal to, greater than, less
than, or any number in between 9, 15, 30, 50, 75, 100, 125, 150,
175, 200, 250, or 300 consecutive nucleotides of the protease
domain of SEQ. ID. Nos. 1 or 35). In some embodiments, the X domain
can comprise the polypeptide encoded by the nucleic acid sequence
of residues 218-1568 of SEQ ID NOs: 1 or 35, or encoded by an
analogous sequence of any NS3/4A nucleic acid, or a fragment
thereof (e.g., said encoding fragment can comprise, consist of, or
consist essentially of about at least, equal to, greater than, less
than, or any number in between 9, 15, 30, 50, 75, 100, 125, 150,
175, 200, 250, or 300 consecutive nucleotides of the helicase
domain of SEQ. ID. Nos. 1 or 35). In some embodiments, the Y domain
can comprise a polypeptide encoded by the nucleic acid sequence of
residues 1569-2069 of SEQ ID NOs: 1 or 35, or encoded by an
analogous sequence of any NS3/4A nucleic acid, or a fragment
thereof (e.g., said fragment can comprise, consist of, or consist
essentially of a polypeptide encoded by about at least, equal to,
greater than, less than, or any number in between 9, 15, 30, 50,
75, 100, 125, 150, 175, 200, 250, 300, or 350 consecutive
nucleotides of the NS4A domain of SEQ. ID. Nos. 1 or 35). In
preferred embodiments, the Z domain can comprise a polypeptide
encoded by the nucleic acid sequence of an antigen, preferably a
TCE, and more preferably a sequence selected from the group
consisting of SEQ ID NOs: 221-571 and SEQ ID NOs: 809-1011 and SEQ
ID NO: 1014, SEQ ID NOs: 1016-1034, SEQ ID NOs: 1146-1173 and SEQ
ID NOs: 1210-1328.
[0100] In some embodiments, the Z domain is located within or
flanking (e.g., juxtaposed or immediately adjacent to) said W
domain. For example, the nucleic acid encoding the Z domain can
place the encoded antigen between or next to amino acids 1 and 2, 2
and 3, 3 and 4, 4 and 5, 5 and 6, 6 and 7, 7 and 8, 8 and 9, 9 and
10, 10 and 11, 11 and 12, 12 and 13, 13 and 14, 14 and 15, 15 and
16, 16 and 17, 17 and 18, 18 and 19, 19 and 20, 20 and 21, 21 and
22, 22 and 23, 23 and 24, 24 and 25, 25 and 26, 26 and 27, 27 and
28, 28 and 29, 29 and 30, 30 and 31, 31 and 32, 32 and 33, 33 and
34, 34 and 35, 35 and 36, 36 and 37, 37 and 38, 38 and 39, 39 and
40, 40 and 41, 41 and 42, 42 and 43, 43 and 44, 44 and 45, 45 and
46, 46 and 47, 47 and 48, 48 and 49, 49 and 50, 50 and 51, 51 and
52, 52 and 53, 53 and 54, 54 and 55, 55 and 56, 56 and 57, 57 and
58, 58 and 59, 59 and 60, 60 and 61, 61 and 62, 62 and 63, 63 and
64, 64 and 65, 65 and 66, 66 and 67, 67 and 68, 68 and 69, 69 and
70, 70 and 71, 71 and 72, 72 and 73, 73 and 74, 74 and 75, 75 and
76, 76 and 77, 77 and 78, 78 and 79, 79 and 80, 80 and 81, 81 and
82, 82 and 83, 83 and 84, 84 and 85, 85 and 86, 86 and 87, 87 and
88, 88 and 89, 89 and 90, 90 and 91, 91 and 92, 92 and 93, 93 and
94, 94 and 95, 95 and 96, 96 and 97, 97 and 98, 98 and 99, 99 and
100, 100 and 101, 101 and 102, 102 and 103, 103 and 104, 104 and
105, 105 and 106, 106 and 107, 107 and 108, 108 and 109, 109 and
110, 110 and 111, 111 and 112, 112 and 113, 113 and 114, 114 and
115, 115 and 116, 116 and 117, 117 and 118, 118 and 119, 119 and
120, 120 and 121, 121 and 122, 122 and 123, 123 and 124, 124 and
125, 125 and 126, 126 and 127, 127 and 128, 128 and 129, 129 and
130, 130 and 131, 131 and 132, 132 and 133, 133 and 134, 134 and
135, 135 and 136, 136 and 137, 137 and 138, 138 and 139, 139 and
140, 140 and 141, 141 and 142, 142 and 143, 143 and 144, 144 and
145, 145 and 146, 146 and 147, 147 and 148, 148 and 149, 149 and
150, 150 and 151, 151 and 152, 152 and 153, 153 and 154, 154 and
155, 155 and 156, 156 and 157, 157 and 158, 158 and 159, 159 and
160, 160 and 161, 161 and 162, 162 and 163, 163 and 164, 164 and
165, 165 and 166, 166 and 167, 167 and 168, 168 and 169, 169 and
170, 170 and 171, 171 and 172, 172 and 173, 173 and 174, 174 and
175, 175 and 176, 176 and 177, 177 and 178, 178 and 179, 179 and
180, or 180 and 181 of said W domain of said chimeric
polypeptide.
[0101] In some embodiments, the Z domain is located within or
flanking (e.g., juxtaposed or immediately adjacent to) the X
domain. For example, the Z domain can place the antigen between or
next to amino acids 1 and 2, 2 and 3, 3 and 4, 4 and 5, 5 and 6, 6
and 7, 7 and 8, 8 and 9, 9 and 10, 10 and 11, 11 and 12, 12 and 13,
13 and 14, 14 and 15, 15 and 16, 16 and 17, 17 and 18, 18 and 19,
19 and 20, 20 and 21, 21 and 22, 22 and 23, 23 and 24, 24 and 25,
25 and 26, 26 and 27, 27 and 28, 28 and 29, 29 and 30, 30 and 31,
31 and 32, 32 and 33, 33 and 34, 34 and 35, 35 and 36, 36 and 37,
37 and 38, 38 and 39, 39 and 40, 40 and 41, 41 and 42, 42 and 43,
43 and 44, 44 and 45, 45 and 46, 46 and 47, 47 and 48, 48 and 49,
49 and 50, 50 and 51, 51 and 52, 52 and 53, 53 and 54, 54 and 55,
55 and 56, 56 and 57, 57 and 58, 58 and 59, 59 and 60, 60 and 61,
61 and 62, 62 and 63, 63 and 64, 64 and 65, 65 and 66, 66 and 67,
67 and 68, 68 and 69, 69 and 70, 70 and 71, 71 and 72, 72 and 73,
73 and 74, 74 and 75, 75 and 76, 76 and 77, 77 and 78, 78 and 79,
79 and 80, 80 and 81, 81 and 82, 82 and 83, 83 and 84, 84 and 85,
85 and 86, 86 and 87, 87 and 88, 88 and 89, 89 and 90, 90 and 91,
91 and 92, 92 and 93, 93 and 94, 94 and 95, 95 and 96, 96 and 97,
97 and 98, 98 and 99, 99 and 100, 100 and 101, 101 and 102, 102 and
103, 103 and 104, 104 and 105, 105 and 106, 106 and 107, 107 and
108, 108 and 109, 109 and 110, 110 and 111, 111 and 112, 112 and
113, 113 and 114, 114 and 115, 115 and 116, 116 and 117, 117 and
118, 118 and 119, 119 and 120, 120 and 121, 121 and 122, 122 and
123, 123 and 124, 124 and 125, 125 and 126, 126 and 127, 127 and
128, 128 and 129, 129 and 130, 130 and 131, 131 and 132, 132 and
133, 133 and 134, 134 and 135, 135 and 136, 136 and 137, 137 and
138, 138 and 139, 139 and 140, 140 and 141, 141 and 142, 142 and
143, 143 and 144, 144 and 145, 145 and 146, 146 and 147, 147 and
148, 148 and 149, 149 and 150, 150 and 151, 151 and 152, 152 and
153, 153 and 154, 154 and 155, 155 and 156, 156 and 157, 157 and
158, 158 and 159, 159 and 160, 160 and 161, 161 and 162, 162 and
163, 163 and 164, 164 and 165, 165 and 166, 166 and 167, 167 and
168, 168 and 169, 169 and 170, 170 and 171, 171 and 172, 172 and
173, 173 and 174, 174 and 175, 175 and 176, 176 and 177, 177 and
178, 178 and 179, 179 and 180, 180 and 181, 181 and 182, 182 and
183, 183 and 184, 184 and 185, 185 and 186, 186 and 187, 187 and
188, 188 and 189, 189 and 190, 190 and 191, 191 and 192, 192 and
193, 193 and 194, 194 and 195, 195 and 196, 196 and 197, 197 and
198, 198 and 199, 199 and 200, 200 and 201, 201 and 202, 202 and
203, 203 and 204, 204 and 205, 205 and 206, 206 and 207, 207 and
208, 208 and 209, 209 and 210, 210 and 211, 211 and 212, 212 and
213, 213 and 214, 214 and 215, 215 and 216, 216 and 217, 217 and
218, 218 and 219, 219 and 220, 220 and 221, 221 and 222, 222 and
223, 223 and 224, 224 and 225, 225, and 226, 226 and 227, 227 and
228, 228 and 229, 229 and 230, 230 and 231, 231 and 232, 232 and
233, 233 and 234, 234 and 235, 235 and 236, 236 and 237, 237 and
238, 238 and 239, 239 and 240, 240 and 241, 241 and 242, 242 and
243, 243 and 244, 244 and 245, 245 and 246, 246 and 247, 247 and
248, 248 and 249, 249 and 250, 250 and 251, 251 and 252, 252 and
253, 253 and 254, 254 and 255, 255 and 256, 256 and 257, 257 and
258, 258 and 259, 259 and 260, 260 and 261, 261 and 262, 262 and
263, 263 and 264, 264 and 265, 265 and 266, 266 and 267, 267 and
268, 268 and 269, 269 and 270, 270 and 271, 271 and 272, 272 and
273, 273 and 274, 274 and 275, 275 and 276, 276 and 277, 277 and
278, 278 and 279, 279 and 280, 280 and 281, 281 and 282, 282 and
283, 283 and 284, 284 and 285, 285 and 286, 286 and 287, 287 and
288, 288 and 289, 289 and 290, 290 and 291, 291 and 292, 292 and
293, 293 and 294, 294 and 295, 295 and 296, 296 and 297, 297 and
298, 298 and 299, 299 and 300, 300 and 201, 301 and 302, 302 and
303, 303 and 304, 304 and 305, 305 and 306, 306 and 307, 307 and
308, 308 and 309, 309 and 310, 310 and 311, 311 and 312, 312 and
313, 313 and 314, 314 and 315, 315 and 316, 316 and 317, 317 and
318, 318 and 319, 319 and 320, 320 and 321, 321, and 322, 322 and
323, 323 and 324, 324 and 325, 325, and 326, 326 and 327, 327 and
328, 328 and 329, 329 and 330, 330 and 331, 331 and 332, 332 and
333, 333 and 334, 334 and 335, 335 and 336, 336 and 337, 337 and
338, 338 and 339, 339 and 340, 340 and 341, 341 and 342, 342 and
343, 343 and 344, 344 and 345, 345 and 346, 346 and 347, 347 and
348, 348 and 349, 349 and 350, 350 and 351, 351 and 352, 352 and
353, 353 and 354, 354 and 355, 355 and 356, 356 and 357, 357 and
358, 358 and 359, 359 and 360, 360 and 361, 361 and 362, 362 and
363, 363 and 364, 364 and 365, 365 and 366, 366 and 367, 367 and
368, 368 and 369, 369 and 370, 370 and 371, 371 and 372, 372 and
373, 373 and 374, 374 and 375, 375 and 376, 376 and 377, 377 and
378, 378 and 379, 379 and 380, 380 and 381, 381 and 382, 382 and
383, 383 and 384, 384 and 385, 385 and 386, 386 and 387, 387 and
388, 388 and 389, 389 and 390, 390 and 391, 391 and 392, 392 and
393, 393 and 394, 394 and 395, 395 and 396, 396 and 397, 397 and
398, 398 and 399, 399 and 400, 401 and 402, 402 and 403, 403 and
404, 404 and 405, 405 and 406, 406 and 407, 407 and 408, 408 and
409, 409 and 410, 410 and 411, 411 and 412, 412 and 413, 413 and
414, 414 and 415, 415 and 416, 416 and 417, 417 and 418, 418 and
419, 419 and 420, 420 and 421, 421 and 422, 422 and 423, 423 and
424, 424 and 425, 425 and 426, 426 and 427, 427 and 428, 428 and
429, 429 and 430, 430 and 431, 431 and 432, 432 and 433, 433 and
434, 434 and 435, 435 and 436, 436 and 437, 437 and 438, 438 and
439, 439 and 440, 440 and 441, 441 and 442, 442 and 443, 443 and
444, 444 and 445, 445 and 446, 446 and 447, 447 and 448, 448 and
449, or 449 and 450 of the X domain of the chimeric polypeptide.
Preferably, the Z domain is located between amino acids 383 and 450
of said X domain.
[0102] Optionally, the Z domain is located within or flanking
(e.g., juxtaposed or immediately adjacent to) said Y domain. For
example, the Z domain can place the antigen between or next to
amino acids 1 and 2, 2 and 3, 3 and 4, 4 and 5, 5 and 6, 6 and 7, 7
and 8, 8 and 9, 9 and 10, 10 and 11, 11 and 12, 12 and 13, 13 and
14, 14 and 15, 15 and 16, 16 and 17, 17 and 18, 18 and 19, 19 and
20, 20 and 21, 21 and 22, 22 and 23, 23 and 24, 24 and 25, 25 and
26, 26 and 27, 27 and 28, 28 and 29, 29 and 30, 30 and 31, 31 and
32, 32 and 33, 33 and 34, 34 and 35, 35 and 36, 36 and 37, 37 and
38, 38 and 39, 39 and 40, 40 and 41, 41 and 42, 42 and 43, 43 and
44, 44 and 45, 45 and 46, 46 and 47, 47 and 48, 48 and 49, 49 and
50, 50 and 51, 51 and 52, 52 and 53, or 53 and 54 of the Y domain
of the chimeric polypeptide.
[0103] That is, aspects of the invention concern a composition that
comprises, consists of, or consists essentially of an isolated
polypeptide provided by the formula:
WXYZ
[0104] wherein:
[0105] W represents a protease domain (e.g., an HCV NS3 protease
domain prepared as described herein, such as a mutant with enhanced
or altered protease activity or substrate specificity) or a
fragment thereof, wherein said fragment comprises, consists of, or
consists essentially of about at least, equal to, greater than,
less than, or any number in between 9, 15, 30, 50, 75, 100, 125,
150, or 175 consecutive amino acids of the protease domain of SEQ.
ID. Nos. 2 or 36 (e.g., residues 1-181 of SEQ ID NO: 36, or an
analogous position in any NS3/4A polypeptide);
[0106] X represents a helicase domain (e.g., an HCV helicase
domain) or a fragment thereof, wherein said fragment comprises,
consists of, or consists essentially of about at least, equal to,
greater than, less than, or any number in between 9, 15, 30, 50,
75, 100, 125, 150, 175, 200, 250, or 300 consecutive amino acids of
the helicase domain of SEQ. ID. Nos. 2 or 36 (e.g., residues 70-596
of SEQ ID NO: 36, or an analogous position in any NS3/4A
polypeptide);
[0107] Y represents an enhancer domain (e.g., an HCV NS4A domain)
or a fragment thereof, wherein said fragment comprises, consists
of, or consists essentially of about at least, equal to, greater
than, less than, or any number in between 9, 15, 30, 50, 75, 100,
125, 150, 175, 200, 250, or 300 consecutive amino acids of the NS4A
domain of SEQ. ID. Nos. 1 or 35 (e.g., residues 597-686 of SEQ ID
NO: 36), or an analogous position in any NS3/4A polypeptide);
and
[0108] Z represents an antigen (e.g., an antigen of a virus,
bacteria, toxin, or cancer cell, such as a TCE provided by a
sequence selected from the group consisting of SEQ ID NOs: 221-571,
SEQ ID NOs: 809-1011, SEQ ID NO: 1014, SEQ ID NOs: 1016-1034, SEQ
ID NOs: 1146-1173, and SEQ ID NOs: 1210-1328), [0109] with the
proviso that Z is not in a position that is naturally occurring in
HCV (e.g., when Z is an HCV antigen, the antigen is inserted at a
position that is not naturally occurring in HCV).
[0110] Aspects of the invention also concern a composition that
comprises, consists of, or consists essentially of an isolated
polypeptide encoded by a nucleic acid provided by the formula:
ZWXY
[0111] wherein:
[0112] W represents a protease domain (e.g., an HCV NS3 protease
domain prepared as described herein, such as a mutant with enhanced
or altered protease activity or substrate specificity) or a
fragment thereof, wherein said fragment comprises, consists of, or
consists essentially of about at least, equal to, greater than,
less than, or any number in between 9, 15, 30, 50, 75, 100, 125,
150, 175, 200, 250, or 300 consecutive nucleotides of the protease
domain of SEQ. ID. Nos. 1 or 35 (e.g., residues 1-551 of SEQ ID NO:
35, or an analogous position in any NS3/4A nucleic acid);
[0113] X represents a helicase domain (e.g., an HCV helicase
domain) or a fragment thereof, wherein said fragment comprises,
consists of, or consists essentially of a polypeptide encoded by
about at least, equal to, greater than, less than, or any number in
between 9, 15, 30, 50, 75, 100, 125, 150, 175, 200, 250, or 300
consecutive nucleotides of the helicase domain of SEQ. ID. Nos. 1
or 35 (e.g., residues 218-1568 of SEQ ID NO: 35, or an analogous
position in any NS3/4A nucleic acid);
[0114] Y represents an enhancer domain (e.g., an HCV NS4A domain)
or a fragment thereof, wherein said fragment comprises, consists
of, or consists essentially of a polypeptide encoded by about at
least, equal to, greater than, less than, or any number in between
9, 15, 30, 50, 75, 100, 125, 150, 175, 200, 250, or 300 consecutive
nucleotides of the NS4A domain of SEQ. ID. Nos. 1 or 35 (e.g.,
residues 1569-2069 of SEQ ID NO: 35, or an analogous position in
any NS3/4A nucleic acid); and
[0115] Z represents an antigen (e.g., an antigen of a virus,
bacteria, toxin, or cancer cell, such as a TCE provided by a
sequence selected from the group consisting of SEQ ID NOs: 221-571,
SEQ ID NOs: 809-1011, SEQ ID NO: 1014, SEQ ID Nos: 1016-1034, SEQ
ID NOs: 1146-1173, and SEQ ID NOs: 1210-1328), [0116] with the
proviso that Z is not in a position that is naturally occurring in
HCV (e.g., when Z is an HCV antigen, the antigen is inserted at a
position that is not naturally occurring in HCV)
[0117] Aspects of the invention also concern a composition that
comprises, consists of, or consists essentially of an isolated
polypeptide encoded by a nucleic acid provided by the formula:
WZXY
[0118] wherein:
[0119] W encodes a protease domain (e.g., an HCV NS3 protease
domain prepared as described herein, such as a mutant with enhanced
or altered protease activity or substrate specificity) or a
fragment thereof, wherein said fragment comprises, consists of, or
consists essentially of about at least, equal to, greater than,
less than, or any number in between 9, 15, 30, 50, 75, 100, 125,
150, 175, 200, 250, or 300 consecutive nucleotides of the protease
domain of SEQ. ID. Nos. 1 or 35 (e.g., residues 1-551 of SEQ ID NO:
35, or an analogous position in any NS3/4A nucleic acid);
[0120] X encodes a helicase domain (e.g., an HCV helicase domain)
or a fragment thereof, wherein said fragment comprises, consists
of, or consists essentially of about at least, equal to, greater
than, less than, or any number in between 9, 15, 30, 50, 75, 100,
125, 150, 175, 200, 250, or 300 consecutive nucleotides of the
helicase domain of SEQ. ID. Nos. 1 or 35 (e.g., residues 218-1568
of SEQ ID NO: 35, or an analogous position in any NS3/4A nucleic
acid);
[0121] Y encodes an enhancer domain (e.g., an HCV NS4A domain) or a
fragment thereof, wherein said fragment comprises, consists of, or
consists essentially of about at least, equal to, greater than,
less than, or any number in between 9, 15, 30, 50, 75, 100, 125,
150, 175, 200, 250, or 300 consecutive nucleotides of the NS4A
domain of SEQ. ID. Nos. 1 or 35 (e.g., residues 1569-2069 of SEQ ID
NO: 35, or an analogous position in any NS3/4A nucleic acid);
and
[0122] Z encodes an antigen (e.g., an antigen of a virus, bacteria,
toxin, or cancer cell, such as a TCE provided by a sequence
selected from the group consisting of SEQ ID NOs: 221-571, SEQ ID
NOs: 809-1011, SEQ ID NO: 1014, SEQ ID NOs: 1016-1034, SEQ ID NOs:
1146-1173, and SEQ ID NOs: 1210-1328), [0123] with the proviso that
Z is not in a position that is naturally occurring in HCV (e.g.,
when Z is an HCV antigen, the antigen is inserted at a position
that is not naturally occurring in HCV).
[0124] Aspects of the invention also concern a composition that
comprises, consists of, or consists essentially of an isolated
polypeptide encoded by a nucleic acid provided by the formula:
WZXY
[0125] wherein:
[0126] W encodes a protease domain (e.g., an HCV NS3 protease
domain prepared as described herein, such as a mutant with enhanced
or altered protease activity or substrate specificity) or a
fragment thereof, wherein said fragment comprises, consists of, or
consists essentially of about at least, equal to, greater than,
less than, or any number in between 9, 15, 30, 50, 75, 100, 125,
150, 175, 200, 250, or 300 consecutive nucleotides of the protease
domain of SEQ. ID. Nos. 1 or 35 (e.g., residues 1-551 of SEQ ID NO:
35, or an analogous position in any NS3/4A nucleic acid);
[0127] X encodes a helicase domain (e.g., an HCV helicase domain)
or a fragment thereof, wherein said fragment comprises, consists
of, or consists essentially of about at least, equal to, greater
than, less than, or any number in between 9, 15, 30, 50, 75, 100,
125, 150, 175, 200, 250, or 300 consecutive nucleotides of the
helicase domain of SEQ. ID. Nos. 1 or 35 (e.g., residues 218-1568
of SEQ ID NO: 35, or an analogous position in any NS3/4A nucleic
acid);
[0128] Y encodes an enhancer domain (e.g., an HCV NS4A domain) or a
fragment thereof, wherein said fragment comprises, consists of, or
consists essentially of about at least, equal to, greater than,
less than, or any number in between 9, 15, 30, 50, 75, 100, 125,
150, 175, 200, 250, or 300 consecutive nucleotides of the NS4A
domain of SEQ. ID. Nos. 1 or 35 (e.g., residues 1569-2069 of SEQ ID
NO: 35, or an analogous position in any NS3/4A nucleic acid);
and
[0129] Z encodes an antigen (e.g., an antigen of a virus, bacteria,
toxin, or cancer cell, such as a TCE provided by a sequence
selected from the group consisting of SEQ ID NOs: 221-571, SEQ ID
NOs: 809-1011, SEQ ID NO: 1014, SEQ ID NOs: 1016-1034, SEQ ID NOs:
1146-1173, and SEQ ID NOs: 1210-1328), [0130] with the proviso that
Z is not in a position that is naturally occurring in HCV (e.g.,
when Z is an HCV antigen, the antigen is inserted at a position
that is not naturally occurring in HCV).
[0131] Aspects of the invention also concern a composition that
comprises, consists of, or consists essentially of an isolated
polypeptide encoded by a nucleic acid provided by the formula:
WXZY
[0132] wherein:
[0133] W encodes a protease domain (e.g., an HCV NS3 protease
domain prepared as described herein, such as a mutant with enhanced
or altered protease activity or substrate specificity) or a
fragment thereof, wherein said fragment comprises, consists of, or
consists essentially of about at least, equal to, greater than,
less than, or any number in between 9, 15, 30, 50, 75, 100, 125,
150, 175, 200, 250, or 300 consecutive nucleotides of the protease
domain of SEQ. ID. Nos. 1 or 35 (e.g., residues 1-551 of SEQ ID NO:
35, or an analogous position in any NS3/4A nucleic acid);
[0134] X encodes a helicase domain (e.g., an HCV helicase domain)
or a fragment thereof, wherein said fragment comprises, consists
of, or consists essentially of about at least, equal to, greater
than, less than, or any number in between 9, 15, 30, 50, 75, 100,
125, 150, 175, 200, 250, or 300 consecutive nucleotides of the
helicase domain of SEQ. ID. Nos. 1 or 35 (e.g., residues 218-1568
of SEQ ID NO: 35, or an analogous position in any NS3/4A nucleic
acid);
[0135] Y encodes an enhancer domain (e.g., an HCV NS4A domain) or a
fragment thereof, wherein said fragment comprises, consists of, or
consists essentially of about at least, equal to, greater than,
less than, or any number in between 9, 15, 30, 50, 75, 100, 125,
150, 175, 200, 250, or 300 consecutive nucleotides of the NS4A
domain of SEQ. ID. Nos. 1 or 35 (e.g., residues 1569-2069 of SEQ ID
NO: 35, or an analogous position in any NS3/4A nucleic acid)
and
[0136] Z encodes an antigen (e.g., an antigen of a virus, bacteria,
toxin, or cancer cell, such as a TCE provided by a sequence
selected from the group consisting of SEQ ID NOs: 221-571, SEQ ID
NOs: 809-1011, SEQ ID NO: 1014, SEQ ID NOs: 1016-1034, SEQ ID NOs:
1146-1173 and SEQ ID NOs: 1210-1328), [0137] with the proviso that
Z is not in a position that is naturally occurring in HCV (e.g.,
when Z is an HCV antigen, the antigen is inserted at a position
that is not naturally occurring in HCV).
[0138] Methods of making and using the compositions described
herein are also provided. In addition to methods of making the
embodied nucleic acids and polypeptides, other embodiments include
method of making immunogens and/or vaccine compositions. Preferably
immunogenic compositions or vaccine compositions comprise a
pharmaceutically acceptable carrier in addition to the nucleic
acids and/or polypeptides described herein. Some methods are
practiced, for example, by mixing an adjuvant with a nucleic acid
or polypeptide as described above, so as to formulate a single
composition (e.g., an immunogenic composition or a vaccine).
Preferred methods involve the mixing of ribavirin with the nucleic
acids or encoded polypeptides described herein. (See e.g., U.S.
Pat. No. 6,680,059, and U.S. Pat. No. 6,858,590, hereby expressly
incorporated by reference in their entirety.)
[0139] Preferred methods of using the compositions described herein
involve providing an animal (e.g., a mammal such as a human or a
domestic animal) in need of an immune response to an antigen with a
sufficient amount of one or more of the nucleic acid and/or
polypeptide compositions described herein, wherein the TCE or
antigen present in the composition is derived from said
antigen/target (e.g., an antigen present on a pathogen, such as a
virus like hepatitis, HIV, or the flu). In some embodiments, the
animal, preferably a mammal, collectively referred to as a subject,
is identified as a subject in need of an immune response to an
antigen or a pathogen comprising an antigen, and said identified
subject is provided a therapeutically effective amount of one or
more of the NS3/4A platforms comprising said antigen. Optionally,
the immune response of said identified subject to said platform
comprising said antigen is measured.
[0140] In still more embodiments, a gene gun or electroporation
device (e.g., Medpulsar.TM.) is used to introduce the nucleic acids
described herein to a mammalian subject in need of an immune
response to an agent, wherein the nucleic acids encode a TCE
specific to the agent. In some embodiments, an amount of an
adjuvant, such as ribavirin, is mixed with the nucleic acid
immunogen prior to delivery. In other embodiments, the nucleic acid
immunogen is provided shortly before or after administration of
ribavirin or at the same site of nucleic acid inoculation. Other
embodiments relate to an isolated nucleic acid comprising a nucleic
acid sequence encoding a TCE, wherein said nucleic acid sequence
encoding said TCE is inserted within a nucleic acid sequence
comprising, consisting essentially of, or consisting of one of the
following SEQ ID NOs: 1, 35, or 573-806, and wherein said isolated
nucleic acid operably encodes a polypeptide that retains catalytic
activity. These embodiments can be delivered using electroporation
methods, as described above, with or without an adjuvant, such as
ribavirin.
[0141] SEQ ID NOs: 1174-1198 correspond to nucleic acid and peptide
constructs of fragments of the HBcAg with or without NS3 protease
cleavage sites joined to the NS3/4A platform. SEQ ID NOs: 1174-1198
initially correspond to all parts of possible constructs presented
therein, including the amino acid sequence for HBcAg, the NS3/4A
protease cleavage site, the NS4A/B protease cleavage site, and the
NS3/4A platform.
[0142] SEQ ID NOs: 1181-1182 correspond to a functional NS3
protease joined to NS4A via a mutant NS3/4A protease cleavage site.
The NS3/4A platform is joined to the HBcAg without any NS3 protease
cleavage sites. SEQ ID NOs: 1181-1182, corresponding to FIG. 1A,
will have an active protease (NS3) that is unable to cleave
itself.
[0143] SEQ ID NOs: 1183-1184 correspond to a mutant non-functional
NS3 protease joined to NS4A via an NS3/4A protease cleavage site.
The NS3/4A platform is joined to the HBcAg without any NS3 protease
cleavage sites. SEQ ID NOs: 1183-1184, corresponding to FIG. 1B,
will have an inactive protease (NS3) that is unable to cleave
itself through the functional protease cleavage site.
[0144] SEQ ID NOs: 1185-1186 correspond to a functional NS3
protease joined to NS4A via an NS3/4A protease cleavage site. The
NS3/4A platform is joined to the HBcAg without any NS3 protease
cleavage sites. SEQ ID NOs: 1185-1186, corresponding to FIG. 1C,
will have an active protease (NS3) that is able to cleave itself
but will not cleave any other portion of the peptide as there are
no other protease cleavage sites available. Accordingly, the
products created by NS3 cleavage include the NS3 protein and the
NS4A joined to the HBcAg.
[0145] SEQ ID NOs: 1187-1188 correspond to a functional NS3
protease joined to NS4A via an NS3/4A protease cleavage site. The
NS3/4A platform is joined to the HBcAg via an NS4A/B protease
cleavage site. SEQ ID NOs: 1187-1188, corresponding to FIG. 1D,
will have an active protease (NS3) that is able to cleave itself
and cleave the NS4A from the HBcAg via the NS4A/B protease cleavage
site. Accordingly, the products created by NS3 cleavage include the
NS3 protein, the NS4A protein, and the HBcAg peptide.
[0146] SEQ ID NOs: 1189-1190 correspond to a functional NS3
protease joined to NS4A via an NS3/4A protease cleavage site. The
NS3/4A platform is joined to the HBcAg via an NS4A/B protease
cleavage site. The HBcAg contains NS3/4A protease cleavage sites
within the peptide, particularly between amino acids 44 and 45,
between amino acids 87 and 88, as well as between amino acids 141
and 142. SEQ ID NOs: 1189-1190, corresponding to FIG. 1E, will have
an active protease (NS3) that is able to cleave itself and cleave
the NS4A from the HBcAg via the NS4A/B protease cleavage site.
Additionally, the NS3 protease will cleave the HBcAg between amino
acids 44 and 45, between amino acids 87 and 88, as well as between
amino acids 141 and 142 via the NS3/4A protease cleavage site.
Accordingly, the products created by NS3 cleavage include the NS3
protein, the NS4A protein, and the fragments of the HBcAg peptide
corresponding to amino acids 1-44, amino acids 45-87, amino acids
88-141, and amino acids 142-183.
[0147] SEQ ID NOs: 1191-1198 correspond to a functional NS3
protease joined to NS4A via an NS3/4A protease cleavage site. The
NS3/4A platform is joined to the HBcAg via an NS4A/B protease
cleavage site. The HBcAg is shuffled with NS3/4A protease cleavage
sites separating the shuffled fragments. SEQ ID NOs: 1191-1198,
corresponding to FIGS. 1F-1I, will have an active protease (NS3)
that is able to cleave itself and cleave the NS4A from the HBcAg
via the NS4A/B protease cleavage site. Additionally, the NS3
protease will cleave the HBcAg shuffled fragments at the site of
the NS3/4A protease cleavage site. Accordingly, the products
created by NS3 cleavage include the NS3 protein, the NS4A protein,
and the fragments of the HBcAg peptide.
[0148] SEQ ID NOs: 1016-1034, SEQ ID NOs: 1146-1173 and SEQ ID NOs:
1210-1328 correspond to antigenic peptide sequences from allergens
and infectious diseases. The peptides represented can be used as
part of an immunogenic composition designed to raise an immune
response against the antigen from which they originate. In another
embodiment, the nucleic acids coding for the antigenic peptide
sequence can be used as part of DNA vaccine designed to raise an
immune response against the antigen from which they originate after
administration.
[0149] SEQ ID NOs: 1122-1145 correspond to antigenic peptide
sequences containing the NS3/4A protease cleavage site inserted at
various sites within the peptide sequence. Although the SEQ ID NOs:
1122-1145 recite only the NS3/4A protease cleavage site, any NS3
protease cleavage site (e.g., NS4A/B protease cleavage site) can be
used. The protease cleavage sites are cleaved by an NS3 peptide,
which can be administered in peptide or nucleic acid form
pre-administration, post-administration, or peri-administration of
the antigenic sequence containing the NS3 protease cleavage
site(s). The protease cleavage sites improve antigen processing
within antigen presenting cells and facilitate a heightened
cell-mediated immune response. Although the antigenic sequences of
SEQ ID NOs: 1122-1145 contain naturally-ordered antigen fragments
separated by an NS3 protease cleavage site, the antigenic fragments
can be ordered randomly as in the shuffled fragments of HBcAg
separated by NS3 protease cleavage sites seen in SEQ ID NOs:
1174-1198.
[0150] SEQ ID NOs: 1098-1121 correspond to the codon-optimized
(human) nucleic acid sequence encoding antigenic peptide sequences
containing the NS3/4A protease cleavage site inserted at various
sites within the peptide sequence. Although SEQ ID NOs: 1098-1121
encode only the NS3/4A protease cleavage site, any NS3 protease
cleavage site (e.g., NS4A/B, NS4B/5A, and NS5A/5B protease cleavage
sites) can be used. The protease cleavage sites are cleaved by an
NS3 peptide, which can be administered as a peptide or nucleic acid
pre-administration, post-administration, or peri-administration of
the codon optimized nucleic acid coding the antigenic sequence
containing the NS3 protease cleavage site(s). The protease cleavage
sites improve antigen processing within antigen presenting cells
and facilitate a heightened cell-mediated immune response. Although
the codon optimized nucleic acids corresponding to SEQ ID NOs:
1098-1121 encode antigenic sequences containing naturally-ordered
antigen fragments separated by an NS3 protease cleavage site, the
antigenic fragments can be ordered randomly as in the shuffled
fragments of HBcAg separated by NS3 protease cleavage sites seen in
SEQ ID NOs: 1174-1198.
[0151] SEQ ID NOs: 1059-1097 correspond to the NS3/4A peptide
linked to antigenic peptide sequences containing NS3/4A protease
cleavage site inserted at various sites within the peptide
sequence. Although SEQ ID NOs: 1059-1097 encode only the NS3/4A
protease cleavage site, any NS3 protease cleavage site (e.g.,
NS4A/B, NS4B/5A, and NS5A/5B protease cleavage sites) can be used.
The protease cleavage sites improve antigen processing within
antigen presenting cells and facilitate a heightened cell-mediated
immune response. Although the antigenic sequences of SEQ ID NOs:
1059-1097 contain naturally-ordered antigen fragments separated by
an NS3 protease cleavage site, the antigenic fragments can be
ordered randomly as in the shuffled fragments of HBcAg separated by
NS3 protease cleavage sites seen in SEQ ID NOs: 1174-1198.
[0152] SEQ ID NOs: 1035-1058 correspond to the codon-optimized
(human) nucleic acid sequence encoding NS3/4A peptide linked to
antigenic peptide sequences containing the NS3/4A protease cleavage
site inserted at various sites within the peptide sequence.
Although SEQ ID NOs: 1035-1058 encode only the NS3/4A protease
cleavage site, any NS3 protease cleavage site (e.g., NS4A/B,
NS4B/5A, and NS5A/5B protease cleavage sites) can be used. The
protease cleavage sites improve antigen processing within antigen
presenting cells and facilitate a heightened cell-mediated immune
response. Although the codon optimized nucleic acids presented in
SEQ ID NOs: 1035-1058 encode antigenic sequences containing
naturally-ordered antigen fragments separated by an NS3 protease
cleavage site, the antigenic fragments can be ordered randomly as
in the shuffled fragments of HBcAg separated by NS3 protease
cleavage sites seen in SEQ ID NOs: 1174-1198.
[0153] SEQ ID NO: 1175 corresponds to the wild type sequence of
HBcAg and SEQ ID NO: 1176 corresponds to the codon optimized
sequence of HBcAg. SEQ ID NOs: 1199-1209 correspond to mutant
NS3/4A peptides having altered substrate specificity.
BRIEF DESCRIPTION OF THE DRAWINGS
[0154] FIGS. 1A-1I depict various constructs containing the NS3/4A
platform and the HBcAg containing NS3 protease cleavage sites.
[0155] FIG. 2 is a graph showing the antibody titer in H-2.sup.d
mice against NS3 as a function of time after the first intra
muscular immunization. Diamonds denote antibody titer in mice
immunized with NS3/4A-pVAX and squares denote antibody titer in
mice immunized with NS3-pVAX.
[0156] FIGS. 3A and 3B show the mean NS3-specific antibody
responses primed by gene gun immunizations with 4 .mu.g
wtNS3/4A-pVAX1 and coNS3/4A-pVAX1, or s.c. injection of 10.sup.7
wtNS3/4A-SFV particles in groups often H-2.sup.d mice (FIG. 3A).
All mice were immunized at weeks zero and four. Values are given as
mean end-point antibody titres (.+-.SD.). Also shown FIG. 3B are
the IgG subclass patterns from groups of five mice immunized twice
with wtNS3/4A-pVAX1 given i.m., coNS3/4A-pVAX1 given i.m. or by
gene gun (gg), and wtNS3/4A-SFV given s.c. Values are given as mean
end-point antibody titres (.+-.SD.). A "**" sign indicates a
statistical difference of p<0.01, a "*" sign indicates a
difference of p<0.05, and NS (not significant) indicates no
statistical difference (Mann-Whitney). Also given is the titer
ratio obtained by dividing the mean endpoint titre of IgG2a
antibodies to NS3 by the mean endpoint titre IgG1 antibodies to
NS3. A high ratio (>3) indicates a Th1-like response and a low
ratio (<0.3) indicates a Th2-like response, whereas values
within a three-fold difference from 1 (0.3 to 3) indicates a mixed
Th1/Th2 response.
[0157] FIG. 4A shows the in vivo protection conferred by one gene
gun immunization of NS3/4A-pVAX1 (4 .mu.g) or MSLF1-pVAX1 (4
.mu.g). Mice were immunized with the respective plasmid and 14 days
later the mice were challenged with an NS3/4A expressing SP2/0 cell
line (approximately 10.sup.6 cells/mouse). Tumor size was then
measured through the skin daily following day 6 post-challenge and
the data plotted. FIG. 4B shows the in vivo protection conferred by
two gene gun immunizations of NS3/4A-pVAX1 (4 .mu.g) or MSLF1-pVAX1
(4 .mu.g). Mice were immunized with the respective plasmid at weeks
zero and week four and, 14 days after the last immunization, the
mice were challenged with an NS3/4A expressing SP2/0 cell line
(approximately 10.sup.6 cells/mouse). Tumor size was then measured
through the skin daily following day 6 post-challenge and the data
plotted.
[0158] FIG. 5 shows the in vivo protection conferred by three gene
gun immunizations of NS3/4A-pVAX1 (4 .mu.g) or MSLF1-pVAX1 (4
.mu.g). Mice were immunized with the respective plasmid at weeks
zero, week four, and week eight and, 14 days after the last
immunization, the mice were challenged with an NS3/4A expressing
SP2/0 cell line (approximately 10.sup.6 cells/mouse). Tumor size
was then measured through the skin daily following day 6
post-challenge and the data plotted.
[0159] FIG. 6A is a graph showing the percentage of specific
CTL-mediated lysis of SP2/0 target cells as a function of the
effector to target ratio. Phosphate Buffered Saline (PBS) was used
as a control immunogen.
[0160] FIG. 6B is a graph showing the percentage specific
CTL-mediated lysis of SP2/0 target cells as a function of the
effector to target ratio. Plasmid NS3/4A-pVAX was used as the
immunogen.
[0161] FIG. 7A is a graph showing the response of naive splenic T
cells that were stimulated with peptide coated RMA-S cells. The
naive splenic T cells were obtained from C57/BL6 mice.
[0162] FIG. 7B is a graph showing the response of splenic T cells
that were restimulated with peptide coated RMA-S cells. The splenic
T cells were obtained from C57/BL6 mice that were provided a single
4 .mu.g dose of MSLF1-pVAX1.
[0163] FIG. 7C is a graph showing the response of splenic T cells
that were restimulated with peptide coated RMA-S cells. The splenic
T cells were obtained from C57/BL6 mice that were provided a single
4 .mu.g dose of NS3/4A-pVAX1.
[0164] FIG. 7D is a graph showing the response of naive splenic T
cells that were stimulated with peptide coated RMA-S cells. The
naive splenic T cells were obtained from C57/BL6 mice.
[0165] FIG. 7E is a graph showing the response of splenic T cells
that were restimulated with peptide coated RMA-S cells. The splenic
T cells were obtained from C57/BL6 mice that were provided two 4
.mu.g doses of MSLF1-pVAX1.
[0166] FIG. 7F is a graph showing the response of splenic T cells
that were restimulated with peptide coated RMA-S cells. The splenic
T cells were obtained from C57/BL6 mice that were provided two 4
.mu.g doses of NS3/4A-pVAX1.
[0167] FIG. 7G is a graph showing the response of naive splenic T
cells that were stimulated with NS3/4A expressing EL-4 cells. The
naive splenic T cells were obtained from C57/BL6 mice.
[0168] FIG. 7H is a graph showing the response of splenic T cells
that were restimulated with NS3/4A expressing EL-4 cells. The
splenic T cells were obtained from C57/BL6 mice that were provided
a single 4 .mu.g dose of MSLF1-pVAX1.
[0169] FIG. 7I is a graph showing the response of splenic T cells
that were restimulated with NS3/4A expressing EL-4 cells. The
splenic T cells were obtained from C57/BL6 mice that were provided
a single 4 .mu.g dose of NS3/4A-pVAX1.
[0170] FIG. 7J is a graph showing the response of naive splenic T
cells that were stimulated with NS3/4A expressing EL-4 cells. The
naive splenic T cells were obtained from C57/BL6 mice.
[0171] FIG. 7K is a graph showing the response of splenic T cells
that were restimulated with NS3/4A expressing EL-4 cells. The
splenic T cells were obtained from C57/BL6 mice that were provided
two 4 .mu.g doses of MSLF1-pVAX1.
[0172] FIG. 7L is a graph showing the response of splenic T cells
that were restimulated with NS3/4A expressing EL-4 cells. The
splenic T cells were obtained from C57/BL6 mice that were provided
two 4 .mu.g doses of NS3/4A-pVAX1.
[0173] FIG. 8A shows a flow cytometric quantification of the
precursor frequency of NS3/4A-specific CD8+ T cells using
peptide-loaded H-2D.sup.b:Ig fusion protein. In a) the mean %
NS3-specific CD8+ T cells from groups of five mice immunized twice
with wtNS3-pVAX1, wtNS3/4A-pVAX1, or coNS3/4A-pVAX1 using gene gun
is shown. A "*" sign indicates a difference of p<0.05, and NS
(not significant) indicates no statistical difference
(Mann-Whitney). FIG. 8B, also shown, represents the raw data from
representative individual mice from the groups listed above (e, f,
and h), as well as from individual mice immunized once with
coNS3/4A-pVAX1 (b) or wtNS3/4A-SFV (c). In (d) and (g),
non-immunized control mice from the different experiments have been
given. In (i) and (j) the splenocytes were restimulated for five
days with the NS3-peptides prior to analysis. A total of
150,000-200,000 data points were collected and the percentage of
CD8+ cells stained for H-2D.sup.b:Ig are indicated in the
parentheses in each dot-plot.
[0174] FIGS. 9A and 9B show the priming of in vitro detectable CTLs
in H-2.sup.b mice by gene gun immunization of the wtNS3-pVAX1,
wtNS3/4A, and coNS3/4A plasmids, or s.c. injection of wtNS3/4A-SFV
particles. Groups of five to 10 H-2.sup.b mice were immunized once
(FIG. 9A) or twice (FIG. 9B). The percent specific lysis
corresponds to the percent lysis obtained with either NS3-peptide
coated RMA-S cells (upper panel in (FIG. 9A) and (FIG. 9B) or
NS3/4A-expressing EL-4 cells (lower panel in a and b) minus the
percent lysis obtained with unloaded or non-transfected EL-4 cells.
Values have been given for effector to target (E:T) cell ratios of
60:1, 20:1 and 7:1. Each line indicates an individual mouse.
[0175] FIG. 10A shows the specificity of tumor inhibiting immune
responses primed by gene gun immunization. Groups of ten C57BL/6
mice were either left untreated or were given two monthly
immunizations with 4 .mu.g of coNS3/4A-pVAX1. Two weeks after last
immunization, mice were injected subcutaneously with the parental
EL-4 cell line or 10.sup.6 NS3/4A-expressing EL-4 cells. Tumor
sizes were measured through the skin at days 6, 7, 10, 11, 12, and
14 after tumour injection. In FIG. 10B the in vivo functional
effector cell population was determined in groups of 10 C57BL/6
mice immunized twice with the coNS3/4A-pVAX1 plasmid using gene
gun. In two groups either CD4+ or CD8+ T cells were depleted by
administration of monoclonal antibodies one week prior to, and
during, challenge with the NS3/4A-expressing EL-4 cell line. Tumor
sizes were measured through the skin at days 5, 6, 8, 11, 13, 14,
and 15 after tumour injection. Values have been given as the mean
tumor size.+-.standard error. A "**" sign indicates a statistical
difference of p<0.01, a "*" sign indicates a difference of
p<0.05, and NS (not significant) indicates no statistical
difference (area under the curve values compared by ANOVA).
[0176] FIG. 11 shows an evaluation of the ability of different
immunogens to prime HCV NS3/4A-specific tumor-inhibiting responses
after a single immunization. Groups of ten C57BL/6 mice were either
left untreated or were given one immunization with the indicated
immunogen (4 .mu.g DNA using gene gun in (a), (b), (c), (g), and
(h); 10.sup.7 SFV particles s.c. in d; 100 .mu.g peptide in CFA
s.c. in (e); and 20 .mu.g rNS3 in CFA s.c. in (f). Two weeks after
last immunization, mice were injected subcutaneously with 10.sup.6
NS3/4A-expressing EL-4 cells. Tumor sizes were measured through the
skin at days 6 to 19 after tumor injection. Values have been given
as the mean tumor size.+-.standard error. In (a) to (e), as a
negative control the mean data from the group immunized with the
empty pVAX plasmid by gene gun has been plotted in each graph. In
(f) to (h) the negative controls were non-immunized mice. Also
given is the p value obtained from the statistical comparison of
the control with each curve using the area under the curve and
ANOVA.
[0177] FIGS. 12A, 12B, and 12C show the comparative efficiency of
gene gun delivered wtNS3/4A-pVAX1 and coNS3/4A-pVAX1 plasmids in
priming tumor inhibiting immune responses. Groups of ten BALB/c
mice were either left untreated or were given one (FIG. 12A), two
(FIG. 12B) or three (FIG. 12C) monthly immunisations with 4 .mu.g
of plasmid. Two weeks after last immunization, mice were injected
subcutaneously with 10.sup.6 NS3/4A-expressing SP2/0 cells. Tumor
sizes were measured through the skin at days 6, 8, 10, 11, 12, 13,
and 14 after tumor injection. Values have been given as the mean
tumor size.+-.standard error. A "**" sign indicates a statistical
difference of p<0.01, a "*" sign indicates a difference of
p<0.05, and NS (not significant) indicates no statistical
difference (area under the curve values compared by ANOVA).
[0178] FIG. 13 shows the effect of therapeutic vaccination with the
coNS3/4A plasmid using the gene gun. Groups of ten C57BL/6 mice
were inoculated with 10.sup.6 NS3/4A-EL4 cells. One group had been
immunized once with 4 .mu.g coNS3/4A DNA using a gene gun two weeks
prior to challenge (positive control), one group was immunized the
same way six days after tumor inoculation, and one group was
immunized 12 days after tumor inoculation. One group was not
immunized (negative control). Tumor sizes were measured through the
skin at days 6, 10, 11, 12, 13, 14, 18, 19, and 20 after tumour
injection. Values have been given as the mean tumor
size.+-.standard error. A "**" sign indicates a statistical
difference of p<0.01, a "*" sign indicates a difference of
p<0.05, and NS (not significant) indicates no statistical
difference (area under the curve values compared by ANOVA).
[0179] FIG. 14 is a graph showing the humoral response to 10 and
100 .mu.g recombinant Hepatitis C virus (HCV) non structural 3
protein (NS3), as determined by mean end point titres, when a
single dose of 1 mg of ribavirin was co-administered.
[0180] FIG. 15 is a graph showing the humoral response to 20 .mu.g
recombinant Hepatitis C virus (HCV) non structural 3 protein (NS3),
as determined by mean end point titres, when a single dose of 0.1,
1.0, or 10 mg of ribavirin was co-administered.
[0181] FIG. 16 is a graph showing the effects of a single dose of 1
mg ribavirin on NS3-specific lymph node proliferative responses, as
determined by in vitro recall responses.
[0182] FIG. 17 shows the location of amino acid residues in the
NS3A protease that affect protease cleavage. Versions of
NS3/NS4A-pVAX were constructed to encode proteins in which each
amino acid of the shown sequence other than the alanine residues
was substituted with an alanine residue. Each alanine residue was
substituted with a glycine residue. The encoded proteins were
analyzed for protease activity. The red color indicates the 16
mutations which resulted in a protein that lacked all protease
activity. The dark blue color indicates the 3 mutations which
resulted in a protein that exhibited greatly enhanced protease
activity compared to wtNS3/NS4A.
[0183] FIG. 18 depicts the protease activity of the NS3 protease
domain in which alanine or glycine was substituted for each
protease-domain residue. Each mutant was tested for protease
activity after translation. The upper band corresponds to the
noncleaved NS3/4A fusion protein and the lower band corresponds to
the free NS3 protein. A single or clearly dominant peak indicates
destroyed or enhanced protease activity, compared with the dual
peak appearance of the wt NS3/4A gene.
[0184] FIGS. 19A and B depict SDS Page gels where IPS-1 cleavage by
particular mutants are visualized.
[0185] FIG. 20A is a graph showing the response of splenic T cells
that were restimulated with peptide coated RMA-S cells. The splenic
T cells were obtained from C57/BL6 mice that were provided a single
100 .mu.g dose of HBcAg-pVAX1 intramuscularly at week 0 and week 4,
as indicated.
[0186] FIG. 20B is a graph showing the response of splenic T cells
that were restimulated with peptide coated RMA-S cells. The splenic
T cells were obtained from C57/BL6 mice that were provided a single
4 .mu.g dose of HBcAg-pVAX1 with a gene gun at week 0 and week 4,
as indicated.
[0187] FIG. 21 shows the location of amino acid residues in the HCV
NS3 protease that affect protease cleavage. Versions of NS3/4A-pVAX
were constructed to encode proteins in which each amino acid of the
shown sequence other than the alanine residues was substituted with
an alanine residue. Each alanine residue was substituted with a
glycine residue. The encoded polypeptides were analyzed for
protease activity. The red color indicates the 16 mutations which
resulted in a protein that lacked all protease activity. The dark
blue color indicates the 3 mutations which resulted in a protein
that exhibited greatly enhanced protease activity compared to
wtNS3/4A.
[0188] FIG. 22 shows the production of IFN-.gamma. by splenocytes
of mice immunized with various DNA and peptide antigens when primed
with various antigens.
[0189] FIG. 23 shows the level of IgE production by mice immunized
with either recombinant birch or a DNA construct containing a
birch-NS3/4A fusion gene.
[0190] FIGS. 24-1 and 2 shows the lysis of peptide loaded RMA-S
cells.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0191] Several of the embodiments described herein concern
compositions and methods that are useful for generating, enhancing,
or improving an immune response to an epitope of a target antigen.
Disclosed herein are compositions relating to genetic constructs
that include sequences from the Hepatitis C virus (HCV) and
sequences encoding T-cell epitopes (TCEs), and methods of
generating or enhancing an immune response using the genetic
constructs and polypeptides encoded by the genetic constructs.
Several embodiments disclosed herein provide nucleic acids that
encode chimeric HCV NS3/4A polypeptides or fragments thereof of at
least 3 amino acids in length. For example, the NS3/4A sequence or
a fragment thereof can comprise at least, equal to, greater than,
or less than, or any number in between 3, 5, 10, 20, 50, 100, 150,
200, 250, 300, 350, 400, 500, 700, 1000, 1200, or 1500 consecutive
amino acids of a natural or synthetic NS3/4A polypeptide (e.g., a
naturally occurring isotype or a codon-optimized or otherwise
modified NS3/4A polypeptide). Exemplary NS3/4a sequences are
disclosed in U.S. Pat. No. 6,960,569, hereby expressly incorporated
by reference in its entirety. Exemplary codon-optimized sequences
are disclosed in U.S. Patent Application Publication No.
2003/0206919, hereby expressly incorporated by reference in its
entirety, and in SEQ ID NOs: 35 and 36. That is, the nucleic acid
encoding the NS3/4A sequence or a fragment thereof can comprise at
least, equal to, greater than, less than, or any number in between
9, 15, 30, 50, 75, 100, 125, 150, 175, 200, 250, 300, 400, 500,
600, 700, 800, 900, 1000, 1200, 1500, or 2000 consecutive
nucleotides of a nucleic acid sequence that encodes a natural or
synthetic NS3/4A polypeptide. Many of these embodiments also
include a nucleic acid that encodes at least one TCE located within
or flanking (e.g., juxtaposed to) the NS3/4A encoding fragment,
such that the TCE is in a non-naturally occurring position. The
encoded polypeptide retains catalytic activity (i.e., NS3 protease
and/or NS3 helicase activity). Embodiments disclosed herein also
provide chimeric NS3/4A polypeptides or fragments thereof of at
least 3 amino acids in length, which include a TCE within or
flanking (e.g., juxtaposed to) the NS3/4A sequences, such that the
TCE is in a non-naturally-occurring position.
[0192] Generally, the generation, enhancement, or improvement of an
immune response refers to an induction of a humoral (antibody)
response and/or a cellular response. Most simply, an increase in
the amount of antigen-specific antibodies (e.g., total IgG) can be
seen by utilizing one or more of the embodiments described herein.
Enhancement of an immune response refers to any statistically
significant change in the level of one or more immune cells (T
cells, B cells, antigen-presenting cells, dendritic cells and the
like) or in the activity of one or more of these immune cells
(cytotoxic T lymphocyte (CTL) activity, helper T lymphocyte (HTL)
activity, cytokine secretion, change in profile of cytokine
secretion). The skilled artisan will readily appreciate that
several methods for establishing whether an immune response is
generated, enhanced, or improved are available. A variety of
methods for detecting the presence and levels of an immune response
are available, for example. (See, e.g., Current Protocols in
Immunology, Ed: John E. Coligan, et al. (2001) John Wiley &
Sons, NY, N.Y.; Current Protocols in Molecular Biology, (2001),
Greene Publ. Assoc. Inc. & John Wiley & Sons, NY, N.Y.;
Ausubel et al. (2001) Current Protocols in Molecular Biology,
Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc., NY,
N.Y.; Sambrook et al. (1989) Molecular Cloning, Second Ed., Cold
Spring Harbor Laboratory, Plainview, N.Y.); Maniatis et al. (1982)
Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, N.Y.;
and elsewhere). Illustrative methods useful in this context include
intracellular cytokine staining (ICS), ELISPOT, proliferation
assays, cytotoxic T cell assays including chromium release or
equivalent assays, and gene expression analysis using any number of
polymerase chain reaction (PCR) or RT-PCR based assays. For
example, the number of CD8.sup.+ T-cells specific for a particular
antigen or TCE can be measured by flow cytometry. (See, e.g.,
Frelin et al. (2004) Gene Therapy 11:522-533). CTL priming can also
be measured in vivo by, for example, a tumor inhibition model, in
which the ability of an animal (e.g., mouse) to inhibit growth of
tumors derived from tumor cells engineered to express the antigen
of interest. Id.
[0193] In some embodiments, generation or enhancement of an immune
response comprises an increase in target-specific CTL activity of
between 1.5 and 5 fold in a subject that is provided a composition
that comprises the nucleic acids or polypeptides disclosed herein
(e.g., in the context of a chimeric NS3/4A nucleic acid or
polypeptide), wherein the TCE is derived from the target, as
compared to the same TCE that is not provided in the context of the
compositions disclosed herein. In some embodiments, an enhancement
of an immune response comprises an increase in target-specific CTL
activity of about 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5,
8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 15, 16, 17, 18, 19,
20, or more fold in a subject that is provided a composition that
comprises a nucleic acid or a polypeptide disclosed herein (e.g.,
in the context of a chimeric NS3/4A nucleic acid or polypeptide),
wherein the TCE is derived from the target, as compared to as
compared to administration of the same TCE that is not provided in
the context of the compositions disclosed herein.
[0194] In other embodiments, an alteration of an immune response
comprises an increase in target-specific HTL activity, such as
proliferation of helper T cells, of between 1.5 and 5 fold in a
subject that is provided a composition that comprises a nucleic
acid or polypeptide disclosed herein (e.g., in the context of a
chimeric NS3/4A nucleic acid or polypeptide), wherein the TCE is
derived from the target, as compared to the same TCE that is not
provided in the context of the compositions disclosed herein. In
some embodiments, alteration of an immune response comprises an
increase in target-specific HTL activity of about 2, 2.5, 3, 3.5,
4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5,
12, 12.5, 15, 16, 17, 18, 19, 20, or more fold in a subject that is
provided a composition that comprises a nucleic acid or polypeptide
disclosed herein (e.g., in the context of a chimeric NS3/4A nucleic
acid or polypeptide), wherein the TCE is derived from the target,
as compared to administration of the same TCE that is not provided
in the context of the compositions disclosed herein. In this
context, an enhancement in HTL activity may comprise an increase as
described above, or decrease, in production of a particular
cytokine, such as interferon-gamma (IFN.gamma.), interleukin-1
(IL-1), IL-2, IL-3, IL-6, IL-7, IL-12, IL-15, tumor necrosis
factor-alpha (TNF.alpha.), granulocyte macrophage
colony-stimulating factor (GM-CSF), granulocyte-colony stimulating
factor (G-CSF), or other cytokine. In this regard, generation or
enhancement of an immune response may comprise a shift from a Th2
type response to a Th1 type response or in certain embodiments a
shift from a Th1 type response, to a Th2 type response. In other
embodiments, the generation or enhancement of an immune response
may comprise the stimulation of a predominantly Th1 or a Th2 type
response.
[0195] In still more embodiments, an increase in the amount of
antibody specific for the antigen (e.g., total IgG) is increased.
Some embodiments, for example, generate an increase in heterologous
target-specific antibody production of between 1.5, 2, 3, 4, or 5
fold in a subject that is provided a composition comprising the
nucleic acids or polypeptides disclosed herein, (e.g., in the
context of a chimeric NS3/4A nucleic acid or polypeptide), wherein
the TCE is derived from the target, as compared to the same TCE
that is not present in the context of the compositions disclosed
herein. In some embodiments, the increase in heterologous
target-specific antibody production is about 2, 2.5, 3, 3.5, 4,
4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5,
12, 12.5, 15, 16, 17, 18, 19, 20, or more fold in a subject that is
provided a composition that comprises a nucleic acid or polypeptide
disclosed herein, (e.g., in the context of a chimeric NS3/4A
nucleic acid or polypeptide), wherein the TCE is derived from the
target, as compared to as compared to administration of the same
TCE that is not present in the context of the compositions
disclosed herein.
[0196] Generation or enhancement of a cellular immune response can
also refer to the frequency of cytotoxic T lymphocytes (CTLs)
specific for a desired antigen that are primed, or the rapidity of
priming of cytotoxic T lymphocytes (CTLs) specific for a desired
antigen, compared to the priming of CTLs specific for the desired
epitope when the epitope is not presented in the context of the
nucleic acids or peptides disclosed herein. The section below
describes several of the NS3/4A sequences that can be used in the
compositions and methods described herein.
[0197] HCV Sequences
[0198] Several embodiments described herein provide genetic
constructs that contain HCV sequences from the NS3/NS4A region of
HCV. The NS3/NS4A region of HCV has been studied extensively. NS3,
due to its limited genetic variability and relatively large size
(631 amino acids) has in itself been studied as an attractive
target for generating immune responses against HCV. (See
Bartenschlager R., et al. (1995) J. Virol. 67:3835-3844; Pang et
al. (2002) EMBO J. 21:1168-1176). The fact that NS3 is a relatively
large protein renders it less likely to exhibit genetic
non-responder status at the T cell level. (See Frelin et al. (2003)
Gene Therapy 10:689-699). Accordingly, it was contemplated that the
NS3 region of HCV is useful in genetic constructs for generating or
enhancing an immune response to an accompanied target antigen
(e.g., in constructs that encode a TCE derived from a
pathogen).
[0199] The catalytic activity of NS3 is known to affect a host's
ability to mount an immune response to HCV. (See, e.g., Foy, et al.
(2005), Proc. Nat. Acad. Sci. USA 102(8): 2986-2991; Meylan, et al.
(2005) Nature 437(20)1167-1172; Li et al. (2005) Proc. Nat. Acad.
Sci. USA 102(8): 2992-2997). Accordingly, embodiments described
herein relate to genetic constructs encoding catalytically active
NS3/4A polypeptide derivatives, or functional fragments thereof, as
well as the polypeptides encoded by the genetic constructs. As used
herein, the term "functional fragment" of a polypeptide refers to a
variant of the polypeptide that is not full-length yet retains
desired attributes, (e.g. NS3A protease and/or helicase activity,
NS4A co-factor activity, or immunogenicity) of the full-length
native sequence.
[0200] The NS3 protein of HCV possesses both protease and helicase
activity. (See Liu, D. et al., (2001) J. Mol. Biol. 314:543-561).
In preferred embodiments, compositions disclosed herein include
sequences that retain NS3 protease and/or helicase activity. In
addition to cleaving the HCV polypeptide, NS3 protease cleaves host
proteins that normally function to activate the host's innate
immune response. (See, e.g., Foy, et al. (2005), Proc. Nat. Acad.
Sci. USA 102(8): 2986-2991; Meylan, et al. (2005) Nature
437(20)1167-1172; Li et al. (2005) Proc. Nat. Acad. Sci. USA
102(8): 2992-2997). Specifically, NS3 has been shown to cleave the
Toll-like receptor 3 adaptor protein TRIF as well as Cardif. Id.
Accordingly, in some embodiments, the NS3/4A nucleic acid sequences
encode a polypeptide that comprises an NS3 protease domain (e.g., a
sequence that exhibits protease activity).
[0201] NS3 protease activity is localized within the first 181
amino acids of the of NS3/4A peptide. (See, Lin, C. et al., (1994)
J. Virol. 68(12):8147-8157). The NS3 protease domain has a
trypsin-like serine proteinase motif and a zinc binding site. (See,
Love, R. (1996) Cell 87:331-342). Three residues, His57, Asp81 and
Ser139 constitute a catalytic triad typical of the trypsin-like
serine proteases that are strictly conserved in all HCV genotype
sequences. Strict conservation of spacing and order of these
residues is also seen. The active site also contains an
oxyanion/stabilization loop. The zinc binding site of NS3 is
located within amino acids Cys97, Cys99, Cys145 and His149. Id. The
zinc binding site is more highly conserved than the active site and
is responsible for stabilizing the structure of the active site.
Id.
[0202] The crystal structure HCV NS3 with an NS4A polypeptide has
been solved. (See Yao, et al. (1999), Structure 7:1353-1363). Thus,
where the NS3 protease domain contains, for example, an alpha-helix
or a beta-sheet structure, in some embodiments, variants or
modified NS3/4A molecules comprise insertions of amino acids that
maintain that specific structure. In addition to the structural
information above, we describe herein experimental results in which
each and every residue in the NS3 protease domain was
systematically mutated and tested for protease activity, thus
providing guidance in relation to NS3/4A variants, such as which
amino acids in the NS3 protease domain are preferably preserved in
embodiments that retain NS3 protease activity, as well as positions
along the protease domain that can tolerate insertions of TCEs
and/or TCEs and linkers, as discussed in more detail below.
[0203] As used herein the phrase "NS3 protease domain" refers to
sequences encoding the NS3 protease domain from any or all HCV
genotypes or isotypes now known or discovered in the future.
Nucleic acids encoding NS3 protease domains include any nucleic
acid, taking into account the degeneracy of the genetic code that
encodes an NS3 protease domain, and also including codon-optimized
NS3 sequences and modified NS3 sequences derived from
naturally-occurring NS3 nucleic acids. Non-limiting examples of
NS3/4A nucleic acid sequences that can be used with the embodiments
described herein include SEQ ID NOs: 1, 35, and 572-808. By way of
example, NS3 helicase domains can comprise nucleic acid residues
1-551 of SEQ ID NO: 35, or analogous residues in any NS3/4A nucleic
acid. SEQ ID NO: 35 is an exemplary codon-optimized sequence of a
nucleic acid encoding an NS3/NS4A protein generated from an HCV
isolate.
[0204] The NS3 helicase domain resides in the C terminal 450 amino
acids of the protein. Yao et al. (1997) Nat. Struct. Biol.
4(6):463-467. The structure of the helicase domain by itself, in
complex with single-stranded DNA, and in the bifunctional
protease-helicase complexes with NS4A has been solved. (Id. and Kim
et al. (1998), Structure 6:89-100). Previous studies have indicated
that the protease domain of NS3 enhances the helicase activity of
NS3. (See, Frick et al. (2004) J Biol Chem. 279(2):1269-1280). The
available structural information above provide guidance as to the
nature of NS3/4A variants, which include substitutions, insertions
and deletions in the NS3 helicase domain that can be made without
perturbing the catalytic activity of the helicase domain, for
example in embodiments that retain NS3 helicase activity.
[0205] As used herein, the phrase "NS3 helicase domain" refers to
sequences encoding an NS3 helicase domain from any or all HCV
genotypes now known or discovered in the future. Nucleic acids
encoding NS3 helicase domains include any nucleic acid, taking into
account the degeneracy of the genetic code that encodes an NS3
polypeptide and also including codon-optimized NS3 helicase
sequences and modified NS3 helicase sequences derived from
naturally-occurring NS3 helicase nucleic acids. Non-limiting
examples of NS3/4A nucleic acid sequences, including sequences of
NS3 helicase domains, are SEQ ID NOs: 1, 35, and 572-808. By way of
example, NS3 helicase domains can comprise nucleic acid residues
218-1568 of SEQ ID NO: 35, or analogous residues in any NS3/4A
nucleic acid. SEQ ID NO: 35 is an exemplary codon-optimized nucleic
acid sequence of an NS3/NS4A peptide generated from an HCV
isolate.
[0206] The NS4 polypeptide of HCV has been shown to increase the
intracellular stability of NS3 and target NS3 to intracellular
membranes, thereby potentially increasing the immunogenicity of
NS3. (See, Wolk, B. et al. (2000). J. Virol. 74:2293-2304). We
recently demonstrated that NS4A gene from HCV is an enhancer that
increases transcription and immunogenicity of an associated gene or
nucleic acid (e.g., NS3). (See, WO 04/048403, which designated the
United States and was published in English, the disclosure of which
is hereby expressly incorporated by reference in its entirety). The
data illustrate that when HCV-1 NS3/4A was transfected into
mammalian cells, vis a vis a eukaryotic expression vector, the
expression level of NS3 was increased compared to the expression
levels of NS3 alone (i.e., without NS4A). Further, immunization
with an NS3/NS4A construct was shown to prime NS3-specific CTLs,
when the construct was provided either i.m. or transdermally.
Accordingly, embodiments disclosed herein include sequences
encoding an NS4A polypeptide, variant, or functional fragment
thereof.
[0207] As used herein, the term "NS4A" refers to either nucleic
acid or amino acid sequences of the NS4A region from any and all
HCV genotypes now known or discovered in the future. Nucleic acids
encoding NS4A include any nucleic acid, taking into account the
degeneracy of the genetic code, that encodes an NS4A domain and
also includes codon-optimized NS4A sequences and modified NS4A
sequences derived from naturally-occurring NS4A nucleic acids.
Non-limiting examples of NS3/4A nucleic acid sequences, including
sequences of NS4 co-factor domains, are SEQ ID NOs: 1, 35, 567-804.
By way of example, NS3 helicase domains can comprise nucleic acid
residues 1569-2069 of SEQ ID NO: 35, or analogous residues in any
NS3/4A nucleic acid. SEQ ID NO: 35 is an exemplary codon-optimized
nucleic acid sequence of an NS3/NS4A peptide generated from an HCV
isolate. SEQ ID NO: 36 is an exemplary codon-optimized amino acid
sequence of an NS3/4A peptide generated from an HCV isolate.
[0208] Current listings of exemplary HCV nucleic acid and
polypeptide sequences, including NS3/NS4A, are publicly available
at the Los Alamos National Laboratories world-wide web site. HCV
NS3/4A nucleic acid sequences (including novel NS3/NS4A regions)
can also be isolated from patients infected with HCV using the
nucleic acids described herein. (See also, Example 1). RNA obtained
from a patient infected with HCV can be reverse transcribed and the
resultant cDNA can be amplified using PCR or another amplification
technique. The primers are preferably obtained from the NS3/4A
sequence of SEQ. ID. NO.: 1.
[0209] For a review of PCR technology, see Molecular Cloning to
Genetic Engineering White, B. A. Ed. in Methods in Molecular
Biology 67: Humana Press, Totowa (1997) and the publication
entitled "PCR Methods and Applications" (1991, Cold Spring Harbor
Laboratory Press). For amplification of mRNAs, it is within the
scope of the invention to reverse transcribe mRNA into cDNA
followed by PCR (RT-PCR); or, to use a single enzyme for both steps
as described in U.S. Pat. No. 5,322,770. Another technique involves
the use of Reverse Transcriptase Asymmetric Gap Ligase Chain
Reaction (RT-AGLCR), as described by Marshall R. L. et al. (PCR
Methods and Applications 4:80-84, 1994).
[0210] Briefly, RNA is isolated, following standard procedures. A
reverse transcription reaction is performed on the RNA using an
oligonucleotide primer specific for the most 5' end of the
amplified fragment as a primer of first strand synthesis. The
resulting RNA/DNA hybrid is then "tailed" with guanines using a
standard terminal transferase reaction. The hybrid is then digested
with RNAse H, and second strand synthesis is primed with a poly-C
primer. Thus, cDNA sequences upstream of the amplified fragment are
easily isolated. For a review of cloning strategies which can be
used see e.g., Sambrook et al., 1989, supra.
[0211] In each of these amplification procedures, primers on either
side of the sequence to be amplified are added to a suitably
prepared nucleic acid sample along with dNTPs and a thermostable
polymerase, such as Taq polymerase, Pfu polymerase, or Vent
polymerase. The nucleic acid in the sample is denatured and the
primers are specifically hybridized to complementary nucleic acid
sequences in the sample. The hybridized primers are then extended.
Thereafter, another cycle of denaturation, hybridization, and
extension is initiated. The cycles are repeated multiple times to
produce an amplified fragment containing the nucleic acid sequence
between the primer sites. PCR has further been described in several
patents including U.S. Pat. Nos. 4,683,195, 4,683,202 and
4,965,188.
[0212] The primers are selected to be substantially complementary
to a portion of the nucleic acid sequence of (SEQ. ID. NO.: 1) that
is unique to this NS3/4A molecule, thereby allowing the sequences
between the primers to be amplified. Preferably, primers can be any
number between at least 16-20, 20-25, or 25-30 nucleotides in
length. The formation of stable hybrids depends on the melting
temperature (Tm) of the DNA. The Tm depends on the length of the
primer, the ionic strength of the solution and the G+C content. The
higher the G+C content of the primer, the higher is the melting
temperature because G:C pairs are held by three H bonds whereas A:T
pairs have only two. The G+C content of the amplification primers
described herein preferably range between 10% and 75%, more
preferably between 35% and 60%, and most preferably between 40% and
55%. The appropriate length for primers under a particular set of
assay conditions can be empirically determined by one of skill in
the art.
[0213] The spacing of the primers relates to the length of the
segment to be amplified. In the context of the embodiments
described herein, amplified segments carrying nucleic acid sequence
encoding HCV peptides can range in size from at least about 25 bp
to the entire length of the HCV genome. Amplification fragments
from 25-1000 bp are typical, fragments from 50-1000 bp are
preferred and fragments from 100-600 bp are highly preferred. It
will be appreciated that amplification primers can be of any
sequence that allows for specific amplification of the NS3/4A
region and can, for example, include modifications such as
restriction sites to facilitate cloning.
[0214] The PCR product can be subcloned and sequenced to ensure
that the amplified sequences represent the sequences of an HCV
peptide. The PCR fragment can then be used to isolate a full length
cDNA clone by a variety of methods. For example, the amplified
fragment can be labeled and used to screen a cDNA library, such as
a bacteriophage cDNA library. Alternatively, the labeled fragment
can be used to isolate genomic clones via the screening of a
genomic library. Additionally, an expression library can be
constructed utilizing cDNA synthesized from, for example, RNA
isolated from an infected patient. In this manner, HCV gene
products can be isolated using standard antibody screening
techniques in conjunction with antibodies raised against the HCV
gene product. (For screening techniques, see, for example, Harlow,
E. and Lane, eds., 1988, Antibodies: A Laboratory Manual, Cold
Spring Harbor Press, Cold Spring Harbor).
[0215] NS3/NS4A Variant Sequences
[0216] A novel nucleic acid and protein corresponding to the NS3/4A
domain of HCV was cloned from a patient infected with HCV (SEQ. ID.
NO.: 1). A Genebank search revealed that the cloned sequence had
the greatest homology to HCV sequences but was only 93% homologous
to the closest HCV relative (accession no AJ 278830). This novel
peptide (SEQ. ID. NO.: 2) and fragments thereof (e.g., SEQ. ID.
NOs.: 14 and 15) that are any number of consecutive amino acids
between at least 3-50 (e.g., 3, 4, 6, 8, 10, 12, 15, 20, 25, 30,
35, 40, 45, or 50 amino acids in length), nucleic acids encoding
these molecules, vectors having said nucleic acids, and cells
having said vectors, nucleic acids, or peptides are embodiments of
the invention. It was also discovered that both the NS3/4A gene
(SEQ. ID. NO.: 1) and corresponding peptide (SEQ. ID. NO.: 2) were
immunogenic in vivo.
[0217] In certain embodiments, the NS3/4A nucleic acids and
polypeptides of the compositions and methods disclosed herein
include variations in nucleotide and/or amino acid sequences,
compared to native NS3/4A sequences and are referred to as NS3/4A
variants. As used herein, the term "native" refers to naturally
occurring HCV sequences (e.g., available HCV isotypes). Variants
may include a substitution, deletion or insertion of one or more
nucleotides, amino acids, or codons encoding the NS3/4A sequences
of the chimeric NS3/4A polypeptides, which results in a change in
the amino acid sequence of the NS3/4A polypeptide, as compared with
the native sequence. Variants can be engineered, for example, using
any of the techniques and guidelines for conservative and
non-conservative mutations set forth, for instance, in U.S. Pat.
No. 5,364,934.
[0218] Mutants of the novel NS3/4A peptide were created. It was
discovered that truncated mutants (e.g., SEQ. ID. NOs.: 12 and 13)
and mutants that lack a proteolytic cleavage site (SEQ. ID. NOs.:
3-11), were also immunogenic in vivo. These novel peptides (SEQ.
ID. NOs.: 3-13) and fragments thereof (e.g., SEQ. ID. NOs.: 16-26)
that are any number of consecutive amino acids between at least
3-50 (e.g., 3, 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, or 50
amino acids in length), nucleic acids encoding these molecules,
vectors having said nucleic acids, and cells having said vectors,
nucleic acids, or peptides are also embodiments of the
invention.
[0219] A codon-optimized nucleic acid encoding NS3/4a was also
created and was found to be immunogenic. The nucleic acid of SEQ.
ID. NO.: 1 was analyzed for codon usage and the sequence was
compared to the codons that are most commonly used in human cells.
Because HCV is a human pathogen, it was unexpected to discover that
the virus had not yet evolved to use codons that are most
frequently found to encode human proteins (e.g., optimal human
codons). A total of 435 nucleotides were replaced to generate the
codon-optimized synthetic NS3/4A nucleic acid. The NS3/4A peptide
encoded by the codon-optimized nucleic acid sequence (SEQ. ID. NO.:
36) was 98% homologous to HCV-1 and contained a total of 15
different amino acids.
[0220] The codon optimized nucleic acid (MSLF1 or coNS3/4A) (SEQ.
ID. NO.: 35) was found to be more efficiently translated in vitro
than the native NS3/4A and that mice immunized with the MSLF1
containing construct generated significantly more NS3/4A specific
antibodies than mice immunized with a wild-type NS3/4A containing
construct. Further, mice immunized with the MSLF1 containing
construct were found to prime NS3-specific CTLs more effectively
and exhibit better in vivo tumor inhibiting immune responses than
mice immunized with wild-type NS3/4A containing constructs.
[0221] NS3/NS4A genes encoding polypeptides with alanine or glycine
substitutions in the serine protease domain of NS3 (i.e., the first
181 amino acids) (SEQ ID NO's: 40 through 220 and 1329-1339) were
found to have altered protease activity compared to the wtNS3/NS4A
polypeptide.
[0222] The peptides and nucleic acids described above are useful as
immunogens, which can be administered alone or in conjunction with
an adjuvant. Preferred embodiments include compositions that
comprise one or more of the nucleic acids and/or peptides described
above with or without an adjuvant. That is, some of the
compositions described herein are prepared with or without an
adjuvant and comprise, consist, or consist essentially of a NS3/4A
peptide (SEQ. ID. NO.: 2 or SEQ. ID. NO.: 36) or fragments thereof
that are any number of consecutive amino acids between at least
3-50 (e.g., 3, 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, or 50
amino acids in length) (e.g., SEQ. ID. NOs.: 14 and 15) or a
nucleic acid encoding one or more of these molecules (e.g., SEQ.
ID. NO.: 35) or a fragment thereof that is any number of
consecutive nucleotides between at least 12-2112 (e.g., 12-15,
15-20, 20-30, 30-50, 50-100, 100-200, 200-500, 500-1000, 1000-1500,
1500-2079, or 1500-2112 consecutive nucleotides in length).
Additional compositions are prepared with or without an adjuvant
and comprise, consist, or consist essentially of one or more of the
NS3/4A mutant peptides (SEQ. ID. NOs.: 3-13) and fragments thereof
that are any number of consecutive amino acids between at least
3-50 (e.g., 3, 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, or 50
amino acids in length). Some of the compositions described herein
are prepared with or without an adjuvant and comprise, consist, or
consist essentially of a mutant NS3/NS4A peptide (e.g., SEQ ID
NO's: 40 through 220 and 1329-1339), or a nucleic acid encoding one
or more of these molecules.
[0223] It was also discovered that compositions comprising
ribavirin and an antigen (e.g., one or more of the previously
described HCV peptides or nucleic acids) enhance and/or facilitate
an animal's immune response to the antigen. That is, it was
discovered that ribavirin is a very effective "adjuvant," which for
the purposes of this disclosure, refers to a material that has the
ability to enhance or facilitate an immune response to a particular
antigen. The adjuvant activity of ribavirin was manifested by a
significant increase in immune-mediated protection against the
antigen, an increase in the titer of antibody raised to the
antigen, and an increase in proliferative T cell responses.
[0224] Accordingly, compositions (e.g., vaccines and other
medicaments) that comprise ribavirin and one or more of the
peptides or nucleic acids described herein are embodiments of the
invention. These compositions can vary according to the amount of
ribavirin, the form of ribavirin, as well as the sequence of the
HCV nucleic acid or peptide.
[0225] Embodiments of the invention also include methods of making
and using the compositions above. Some methods involve the making
of nucleic acids encoding NS3/4A, codon-optimized NS3/4A, mutant
NS34A, fragments thereof that are any number of consecutive
nucleotides between at least 9-100 (e.g., 9, 12, 15, 18, 21, 24,
27, 30, 50, 60, 75, 80, 90, or 100 consecutive nucleotides in
length), peptides corresponding to said nucleic acids, constructs
comprising said nucleic acids, and cells containing said
compositions. Preferred methods, however, concern the making of
vaccine compositions or immunogenic preparations that comprise,
consist, or consist essentially of the newly discovered NS3/4A
fragment, codon-optimized NS3/4A, or an NS3/4A mutant (e.g., a
truncated mutant, a mutant lacking a proteolytic cleavage site, or
a mutant having altered protease activity), or a fragment thereof
or a nucleic acid encoding one or more of these molecules, as
described above. Preferred fragments for use with the methods
described herein include SEQ. ID. NOs.: 12-27 and fragments of SEQ.
ID. NO.: 35 that contain at least 30 consecutive nucleotides. The
compositions described above can be made by providing an adjuvant
(e.g., ribavirin), providing an HCV antigen (e.g., a peptide
comprising an HCV antigen such as (SEQ. ID. NOs.: 2-11, 36, or
40-220) or a fragment thereof such as, SEQ. ID. NOs.: 12-26 or a
nucleic acid encoding one or more of said peptides), and mixing
said adjuvant and said antigen so as to formulate a composition
that can be used to enhance or facilitate an immune response in a
subject to said antigen.
[0226] Methods of enhancing or promoting an immune response in an
animal, including humans, to an antigen are also provided. Such
methods can be practiced, for example, by identifying an animal in
need of an immune response to HCV and providing said animal a
composition comprising one or more of the nucleic acids or peptides
above and an amount of adjuvant that is effective to enhance or
facilitate an immune response to the antigen/epitope. In some
embodiments, the antigen and the adjuvant are administered
separately, instead of in a single mixture. Preferably, in this
instance, the adjuvant is administered a short time before or a
short time after administering the antigen. Preferred methods
involve providing the animal in need with ribavirin and NS3/4A
(e.g., SEQ. ID. NO.: 2), codon-optimized NS3/4A (e.g., SEQ. ID.
NO.: 36), a mutant NS3/4A (e.g., SEQ. ID. NOs.: 3-13 or 40-220), a
fragment thereof (e.g., SEQ. ID. NOs.: 14-26) containing any number
of consecutive amino acids between at least 3-50 (e.g., 3, 4, 6, 8,
10, 12, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length) or
a nucleic acid encoding any one or more of said molecules.
[0227] Other embodiments concern methods of treating and preventing
HCV infection. By one approach, an immunogen comprising one or more
of the HCV nucleic acids or peptides described herein are used to
prepare a medicament for the treatment and/or prevention of HCV
infection. By another approach, an individual in need of a
medicament that prevents and/or treats HCV infection is identified
and said individual is provided a medicament comprising ribavirin
and an HCV antigen such as NS3/4A (e.g., SEQ. ID. NO.: 2),
codon-optimized NS3/4A (e.g., SEQ. ID. NO.: 36), or a mutant NS3/4A
(e.g., SEQ. ID. NOs.: 3-13 or 40-220), a fragment thereof (e.g.,
SEQ. ID. NOs.: 14-26) containing any number of consecutive amino
acids between at least 3-50 (e.g., 3, 4, 6, 8, 10, 12, 15, 20, 25,
30, 35, 40, 45, or 50 amino acids in length) or a nucleic acid
encoding any one or more of these molecules.
[0228] The section below discusses the discovery of the novel
NS3/4A gene, the codon-optimized NS3/4A gene, the creation of the
NS3/4A mutants, and the characterization of the nucleic acids and
peptides corresponding thereto.
NS3/4A, NS3/4A Mutants, and Codon-Optimized NS3/4A
[0229] A novel nucleic acid and protein corresponding to the NS3/4A
domain of HCV was cloned from a patient infected with HCV (SEQ. ID.
NOs.: 1 and 2). A Genebank search revealed that the cloned sequence
had the greatest homology to HCV sequences but was only 93%
homologous to the closest HCV relative (accession no AJ 278830). A
truncated mutant of the novel NS3/4A peptide and NS3/4A mutants,
which lack a proteolytic cleavage site, (as well as corresponding
nucleic acids) were also created. Further, a human codon-optimized
NS3/4A nucleic acid and peptide were created. It was discovered
that these novel peptides and nucleic acids encoding said peptides
were potent immunogens that can be mixed with adjuvants so as to
make a composition that induces a recipient to provide an immune
response to HCV. The cloning of the novel NS3/4A gene and the
creation of the various NS3/4A mutants and codon optimized NS3/4A
gene are described in the following example.
Example 1
[0230] The NS3/4A sequence was amplified from the serum of an
HCV-infected patient (HCV genotype 1a) using the Polymerase Chain
Reaction (PCR). Total RNA was extracted from serum, and cDNA
synthesis and PCR were performed according to standard protocols
(Chen M et al., J. Med. Virol. 43:223-226 (1995)). The cDNA
synthesis was initiated using the antisense primer "NS4KR" (5'-CCG
TCT AGA TCA GCA CTC TTC CAT TTC ATC-3' (SEQ. ID. NO.: 28)). From
this cDNA, a 2079 base pair DNA fragment of HCV, corresponding to
amino acids 1007 to 1711, which encompasses the NS3 and NS4A genes,
was amplified. A high fidelity polymerase (Expand High Fidelity
PCR, Boehringer-Mannheim, Mannheim, Germany) was used with the
"NS3KF" primer (5'-CCT GAA TTC ATG GCG CCT ATC ACG GCC TAT-3' (SEQ.
ID. NO.: 29) and the NS4KR primer. The NS3KF primer contained a
EcoRI restriction enzyme cleavage site and a start codon and the
primer NS4KR contained a XbaI restriction enzyme cleavage site and
a stop codon.
[0231] The amplified fragment was then sequenced (SEQ. ID. NO.: 1).
Sequence comparison analysis revealed that the gene fragment was
amplified from a viral strain of genotype 1a. A computerized BLAST
search against the Genebank database using the NCBI website
revealed that the closest HCV homologue was 93% identical in
nucleotide sequence.
[0232] The amplified DNA fragment was then digested with EcoRI and
XbaI, and was inserted into a pcDNA3.1/His plasmid (Invitrogen)
digested with the same enzymes. The NS3/4A-pcDNA3.1 plasmid was
then digested with EcoRI and XbaI and the insert was purified using
the QiaQuick kit (Qiagen, Hamburg, Germany) and was ligated to a
EcoRI/XbaI digested pVAX vector (Invitrogen) so as to generate the
NS3/4A-pVAX plasmid.
[0233] The rNS3 truncated mutant was obtained by deleting NS4A
sequence from the NS3/4A DNA. Accordingly, the NS3 gene sequence of
NS3/4A-pVAX was PCR amplified using the primers NS3KF and 3'NotI
(5'-CCA CGC GGC CGC GAC GAC CTA CAG-3' (SEQ. ID. NO.: 30))
containing EcoRI and Not I restriction sites, respectively. The NS3
fragment (1850 bp) was then ligated to a EcoRI and Not I digested
pVAX plasmid to generate the NS3-pVAX vector. Plasmids were grown
in BL21 E. coli cells. The plasmids were sequenced and were
verified by restriction cleavage and the results were as to be
expected based on the original sequence.
[0234] In some embodiments, nucleic acid sequences comprising,
consisting essentially of, or consisting of sequences encoding TCEs
are inserted within or flanking (e.g., juxtaposed to) the
NS3/4A-encoding sequence described herein. In some embodiments, a
linker or adjuvant sequence is also, optionally, inserted within or
flanking (e.g., juxtaposed to) an NS3/4A native or variant
sequence, or a native or variant TCE sequence. For example, the
chimeric NS3/4A polypeptide encoded by the nucleic acids above can
include sequences encoding a TCE, or a TCE flanked on one or both
sides by linkers and/or adjuvant sequences, inserted between amino
acids 1 and 2, 2 and 3, 3 and 4, 4 and 5, 5 and 6, 6 and 7, 7 and
8, 8 and 9, 9 and 10, 10 and 11, 11 and 12, 12 and 13, 13 and 14,
14 and 15, 15 and 16, 16 and 17, 17 and 18, 18 and 19, 19 and 20,
20 and 21, 21 and 22, 22 and 23, 23 and 24, 24 and 25, 25 and 26,
26 and 27, 27 and 28, 28 and 29, 29 and 30, 30 and 31, 31 and 32,
32 and 33, 33 and 34, 34 and 35, 35 and 36, 36 and 37, 37 and 38,
38 and 39, 39 and 40, 40 and 41, 41 and 42, 42 and 43, 43 and 44,
44 and 45, 45 and 46, 46 and 47, 47 and 48, 48 and 49, 49 and 50,
50 and 51, 51 and 52, 52 and 53, 53 and 54, 54 and 55, 55 and 56,
56 and 57, 57 and 58, 58 and 59, 59 and 60, 60 and 61, 61 and 62,
62 and 63, 63 and 64, 64 and 65, 65 and 66, 66 and 67, 67 and 68,
68 and 69, 69 and 70, 70 and 71, 71 and 72, 72 and 73, 73 and 74,
74 and 75, 75 and 76, 76 and 77, 77 and 78, 78 and 79, 79 and 80,
80 and 81, 81 and 82, 82 and 83, 83 and 84, 84 and 85, 85 and 86,
86 and 87, 87 and 88, 88 and 89, 89 and 90, 90 and 91, 91 and 92,
92 and 93, 93 and 94, 94 and 95, 95 and 96, 96 and 97, 97 and 98,
98 and 99, 99 and 100, 100 and 101, 101 and 102, 102 and 103, 103
and 104, 104 and 105, 105 and 106, 106 and 107, 107 and 108, 108
and 109, 109 and 110, 110 and 111, 111 and 112, 112 and 113, 113
and 114, 114 and 115, 115 and 116, 116 and 117, 117 and 118, 118
and 119, 119 and 120, 120 and 121, 121 and 122, 122 and 123, 123
and 124, 124 and 125, 125 and 126, 126 and 127, 127 and 128, 128
and 129, 129 and 130, 130 and 131, 131 and 132, 132 and 133, 133
and 134, 134 and 135, 135 and 136, 136 and 137, 137 and 138, 138
and 139, 139 and 140, 140 and 141, 141 and 142, 142 and 143, 143
and 144, 144 and 145, 145 and 146, 146 and 147, 147 and 148, 148
and 149, 149 and 150, 150 and 151, 151 and 152, 152 and 153, 153
and 154, 154 and 155, 155 and 156, 156 and 157, 157 and 158, 158
and 159, 159 and 160, 160 and 161, 161 and 162, 162 and 163, 163
and 164, 164 and 165, 165 and 166, 166 and 167, 167 and 168, 168
and 169, 169 and 170, 170 and 171, 171 and 172, 172 and 173, 173
and 174, 174 and 175, 175 and 176, 176 and 177, 177 and 178, 178
and 179, 179 and 180, 180 and 181, 181 and 182, 182 and 183, 183
and 184, 184 and 185, 185 and 186, 186 and 187, 187 and 188, 188
and 189, 189 and 190, 190 and 191, 191 and 192, 192 and 193, 193
and 194, 194 and 195, 195 and 196, 196 and 197, 197 and 198, 198
and 199, 199 and 200, 200 and 201, 201 and 202, 202 and 203, 203
and 204, 204 and 205, 205 and 206, 206 and 207, 207 and 208, 208
and 209, 209 and 210, 210 and 211, 211 and 212, 212 and 213, 213
and 214, 214 and 215, 215 and 216, 216 and 217, 217 and 218, 218
and 219, 219 and 220, 220 and 221, 221 and 222, 222 and 223, 223
and 224, 224 and 225, 225, and 226, 226 and 227, 227 and 228, 228
and 229, 229 and 230, 230 and 231, 231 and 232, 232 and 233, 233
and 234, 234 and 235, 235 and 236, 236 and 237, 237 and 238, 238
and 239, 239 and 240, 240 and 241, 241 and 242, 242 and 243, 243
and 244, 244 and 245, 245 and 246, 246 and 247, 247 and 248, 248
and 249, 249 and 250, 250 and 251, 251 and 252, 252 and 253, 253
and 254, 254 and 255, 255 and 256, 256 and 257, 257 and 258, 258
and 259, 259 and 260, 260 and 261, 261 and 262, 262 and 263, 263
and 264, 264 and 265, 265 and 266, 266 and 267, 267 and 268, 268
and 269, 269 and 270, 270 and 271, 271 and 272, 272 and 273, 273
and 274, 274 and 275, 275 and 276, 276 and 277, 277 and 278, 278
and 279, 279 and 280, 280 and 281, 281 and 282, 282 and 283, 283
and 284, 284 and 285, 285 and 286, 286 and 287, 287 and 288, 288
and 289, 289 and 290, 290 and 291, 291 and 292, 292 and 293, 293
and 294, 294 and 295, 295 and 296, 296 and 297, 297 and 298, 298
and 299, 299 and 300, 300 and 201, 301 and 302, 302 and 303, 303
and 304, 304 and 305, 305 and 306, 306 and 307, 307 and 308, 308
and 309, 309 and 310, 310 and 311, 311 and 312, 312 and 313, 313
and 314, 314 and 315, 315 and 316, 316 and 317, 317 and 318, 318
and 319, 319 and 320, 320 and 321, 321, and 322, 322 and 323, 323
and 324, 324 and 325, 325, and 326, 326 and 327, 327 and 328, 328
and 329, 329 and 330, 330 and 331, 331 and 332, 332 and 333, 333
and 334, 334 and 335, 335 and 336, 336 and 337, 337 and 338, 338
and 339, 339 and 340, 340 and 341, 341 and 342, 342 and 343, 343
and 344, 344 and 345, 345 and 346, 346 and 347, 347 and 348, 348
and 349, 349 and 350, 350 and 351, 351 and 352, 352 and 353, 353
and 354, 354 and 355, 355 and 356, 356 and 357, 357 and 358, 358
and 359, 359 and 360, 360 and 361, 361 and 362, 362 and 363, 363
and 364, 364 and 365, 365 and 366, 366 and 367, 367 and 368, 368
and 369, 369 and 370, 370 and 371, 371 and 372, 372 and 373, 373
and 374, 374 and 375, 375 and 376, 376 and 377, 377 and 378, 378
and 379, 379 and 380, 380 and 381, 381 and 382, 382 and 383, 383
and 384, 384 and 385, 385 and 386, 386 and 387, 387 and 388, 388
and 389, 389 and 390, 390 and 391, 391 and 392, 392 and 393, 393
and 394, 394 and 395, 395 and 396, 396 and 397, 397 and 398, 398
and 399, 399 and 400, 401 and 402, 402 and 403, 403 and 404, 404
and 405, 405 and 406, 406 and 407, 407 and 408, 408 and 409, 409
and 410, 410 and 411, 411 and 412, 412 and 413, 413 and 414, 414
and 415, 415 and 416, 416 and 417, 417 and 418, 418 and 419, 419
and 420, 420 and 421, 421 and 422, 422 and 423, 423 and 424, 424
and 425, 425 and 426, 426 and 427, 427 and 428, 428 and 429, 429
and 430, 430 and 431, 431 and 432, 432 and 433, 433 and 434, 434
and 435, 435 and 436, 436 and 437, 437 and 438, 438 and 439, 439
and 440, 440 and 441, 441 and 442, 442 and 443, 443 and 444, 444
and 445, 445 and 446, 446 and 447, 447 and 448, 448 and 449, 449
and 450, 450 and 451, 451 and 452, 452 and 453, 453 and 454, 454
and 455, 455 and 456, 456 and 457, 457 and 458, 458 and 459, 459
and 460, 460 and 461, 461 and 462, 462 and 463, 463 and 464, 464
and 465, 465 and 466, 466 and 467, 467 and 468, 468 and 469, 469
and 470, 470 and 471, 471 and 472, 472 and 473, 473 and 474, 474
and 475, 475 and 476, 476 and 477, 477 and 478, 478 and 479, 479
and 480, 480 and 481, 481 and 482, 482 and 483, 483 and 484, 484
and 485, 485 and 486, 486 and 487, 487 and 488, 488 and 489, 489
and 490, 490 and 491, 491 and 492, 492 and 493, 493 and 494, 494
and 495, 495 and 496, 496 and 497, 497 and 498, 498 and 499, 499
and 500, 501 and 502, 502 and 503, 503 and 504, 504 and 505, 505
and 506, 506 and 507, 507 and 508, 508 and 509, 509 and 510, 510
and 511, 511 and 512, 512 and 513, 513 and 514, 514 and 515, 515
and 516, 516 and 517, 517 and 518, 518 and 519, 519 and 520, 520
and 521, 521 and 522, 522 and 523, 523 and 524, 524 and 525, 525
and 526, 526 and 527, 527 and 528, 528 and 529, 529 and 530, 530
and 531, 531 and 532, 532 and 533, 533 and 534, 534 and 535, 535
and 536, 536 and 537, 537 and 538, 538 and 539, 539 and 540, 540
and 541, 541 and 542, 542 and 543, 543 and 544, 544 and 545, 545
and 546, 546 and 547, 547 and 548, 548 and 549, 549 and 550, 550
and 551, 551 and 552, 552 and 553, 553 and 554, 554 and 555, 555
and 556, 556 and 557, 557 and 558, 558 and 559, 559 and 560, 560
and 561, 561 and 562, 562 and 563, 563 and 564, 564 and 565, 565
and 566, 566 and 567, 567 and 568, 568 and 569, 569 and 570, 570
and 571, 571 and 572, 572 and 573, 573 and 574, 574 and 575, 575
and 576, 576 and 577, 577 and 578, 578 and 579, 579 and 580, 580
and 581, 581 and 582, 582 and 583, 583 and 584, 584 and 585, 585
and 586, 586 and 587, 587 and 588, 588 and 589, 589 and 590, 590
and 591, 591 and 592, 592 and 593, 593 and 594, 594 and 595, 595
and 596, 596 and 597, 597 and 598, 598 and 599, 599 and 600, 601
and 602, 602 and 603, 603 and 604, 604 and 605, 605 and 606, 606
and 607, 607 and 608, 608 and 609, 609 and 610, 610 and 611, 611
and 612, 612 and 613, 613 and 614, 614 and 615, 615 and 616, 616
and 617, 617 and 618, 618 and 619, 619 and 620, 620 and 621, 621
and 622, 622 and 623, 623 and 624, 624 and 625, 625 and 626, 626
and 627, 627 and 628, 628 and 629, 629 and 630, 630 and 631, 631
and 632, 632 and 633, 633 and 634, 634 and 635, 635 and 636, 636
and 637, 637 and 638, 638 and 639, 639 and 640, 640 and 641, 641
and 642, 642 and 643, 643 and 644, 644 and 645, 645 and 646, 646
and 647, 647 and 648, 648 and 649, 649 and 650, 650 and 651, 651
and 652, 652 and 653, 653 and 654, 654 and 655, 655 and 656, 656
and 657, 657 and 658, 658 and 659, 659 and 660, 660 and 661, 661
and 662, 662 and 663, 663 and 664, 664 and 665, 665 and 666, 666
and 667, 667 and 668, 668 and 669, 669 and 670, 670 and 671, 671
and 672, 72 and 673, 673 and 674, 674 and 675, 675 and 676, 676 and
677, 677 and 678, 678 and 679, 679 and 680, 680 and 681, 681 and
682, 682 and 683, 683 and 684, 684 and 685, or 685 and 686 of a
variant NS3/4A polypeptide (e.g., SEQ ID NO: 36). For example, in
preferred embodiments, the chimeric NS3/4A polypeptide encoded by
the nucleic acids above can include sequences encoding a TCE, or a
TCE flanked on one or both sides by linkers and/or adjuvant
sequences, inserted between amino acids 453-513 of SEQ ID NO: 36,
or in an analogous position in any NS3/4A polypeptide. Embodiments
also relate to the polypeptides encoded by said nucleic acids.
[0235] Accordingly, in some embodiments a nucleic acid encoding a
TCE or a TCE and a linker(s) is inserted between the codons of an
NS3/4A-encoding nucleic acid sequence. For example, in some
embodiments, a nucleic acid encoding a TCE or a TCE and a linker(s)
and/or an adjuvant sequence is inserted between nucleotides 3 and
4, 6 and 7, 9 and 10, 12 and 13, 15 and 16, 18 and 19, 21 and 22,
24 and 25, 27 and 28, 30 and 31, 33 and 34, 36 and 37, 39 and 40,
42 and 43, 45 and 46, 48 and 49, 51 and 52, 54 and 55, 57 and 58,
60 and 61, 63 and 64, 66 and 67, 69 and 70, 72 and 73, 75 and 76,
78 and 79, 81 and 82, 84 and 85, 87 and 88, 90 and 91, 93 and 94,
96 and 97, 99 and 100, 102 and 103, 105 and 106, 108 and 109, 111
and 112, 114 and 115, 117 and 118, 120 and 121, 123 and 124, 126
and 127, 129 and 130, 132 and 133, 125 and 136, 138 and 139, 141
and 142, 144 and 145, 147 and 148, 150 and 151, 153 and 154, 156
and 157, 159 and 160, 162 and 163, 165 and 166, 168 and 169, 171
and 172, 174 and 175, 177 and 178, 180 and 181, 183 and 184, 186
and 187, 189 and 19, 192 and 193, 195 and 196, 198 and 199, 201 and
202, 204 and 205, 207 and 208, 210 and 211, 213 and 214, 216 and
217, 219 and 220, 222 and 223, 225 and 226, 228 and 229, 231 and
232, 234 and 235, 237 and 238, 240 and 241, 243 and 244, 246 and
247, 249 and 250, 252 and 253, 255 and 256, 258 and 259, 261 and
262, 264 and 265, 267 and 268, 270 and 271, 273 and 274, 276 and
277, 279 and 280, 282 and 283, 285 and 286, 288 and 289, 291 and
292, 294 and 295, 297 and 298, 300 and 301, 303 and 304, 306 and
307, 309 and 310, 312 and 313, 315 and 316, 318 and 319, 321 and
322, 324 and 325, 327 and 328, 330 and 331, 333 and 334, 336 and
337, 339 and 340, 342 and 343, 345 and 346, 348 and 349, 351 and
352, 354 and 355, 357 and 358, 360 and 361, 363 and 364, 366 and
367, 369 and 370, 372 and 373, 375 and 376, 378 and 379, 381 and
382, 24 and 385, 387 and 388, 390 and 391, 393 and 394, 396 and
397, 399 and 400, 402 and 403, 405 and 406, 408 and 409, 411 and
412, 414 and 415, 417 and 418, 420 and 421, 423 and 424, 426 and
427, 429 and 430, 432 and 433, 435 and 436, 438 and 439, 441 and
442, 444 and 445, 447 and 448, 450 and 451, 453 and 454, 456 and
457, 459 and 460, 462 and 463, 465 and 466, 468 and 469, 471 and
472, 474 and 475, 477 and 478, 480 and 481, 483 and 484, 486 and
487, 489 and 490, 492 and 493, 495 and 496, 498 and 499, 501 and
502, 504 and 505, 507 and 508, 510 and 511, 513 and 514, 516 and
517, 519 and 520, 522 and 523, 525 and 526, 528 and 529, 531 and
532, 534 and 535, 537 and 538, 540 and 541, 543 and 544, 546 and
547, 549 and 550, 552 and 553, 555 and 556, 558 and 559, 561 and
562, 564 and 565, 567 and 568, 570 and 571, 573 and 574, 576 and
577, 579 and 580, 582 and 583, 585 and 586, 588 and 589, 591 and
592, 594 and 595, 597 and 598, 600 and 601, 603 and 604, 606 and
607, 609 and 610, 612 and 613, 615 and 616, 618 and 619, 621 and
622, 624 and 625, 627 and 628, 630 and 631, 633 and 634, 636 and
637, 639 and 640, 642 and 643, 645 and 646, 648 and 649, 651 and
652, 654 and 655, 657 and 658, 660 and 661, 663 and 664, 666 and
667, 669 and 670, 672 and 673, 675 and 676, 678 and 679, 681 and
682, 684 and 685, 687 and 688, 690 and 691, 693 and 694, 696 and
697, 699 and 700, 702 and 703, 705 and 706, 708 and 709, 711 and
712, 714 and 715, 717 and 718, 720 and 721, 723 and 724, 726 and
727, 729 and 730, 732 and 733, 735 and 736, 738 and 739, 741 and
742, 744 and 745, 747 and 748, 750 and 751, 753 and 754, 756 and
757, 759 and 760, 762 and 763, 765 and 766, 768 and 769, 771 and
772, 774 and 775, 777 and 778, 780 and 781, 783 and 784, 786 and
787, 789 and 790, 792 and 793, 795 and 796, 798 and 799, 801 and
802, 804 and 805, 807 and 808, 810 and 811, 813 and 814, 816 and
817, 819 and 820, 822 and 823, 825 and 826, 828 and 829, 831 and
832, 834 and 835, 837 and 838, 840 and 841, 843 and 844, 846 and
847, 849 and 850, 852 and 853, 855 and 856, 858 and 859, 861 and
862, 864 and 865, 867 and 868, 870 and 871, 873 and 874, 876 and
877, 879 and 880, 882 and 883, 885 and 886, 888 and 889, 891 and
892, 894 and 895, 897 and 898, 900 and 901, 903 and 904, 906 and
907, 909 and 910, 912 and 913, 915 and 916, 918 and 919, 921 and
922, 924 and 925, 927 and 928, 930 and 931, 933 and 934, 936 and
937, 939 and 940, 942 and 943, 945 and 946, 948 and 949, 951 and
952, 954 and 955, 957 and 958, 960 and 961, 963 and 964, 966 and
967, 969 and 970, 972 and 973, 975 and 976, 978 and 979, 981 and
982, 984 and 985, 987 and 988, 990 and 991, 993 and 994, 996 and
997, 999 and 1000, 1002 and 1003, 1005 and 1006, 1008 and 1009,
1011 and 1012, 1014 and 1015, 1017 and 1018, 1020 and 1021, 1023
and 1024, 1026 and 1027, 1029 and 1030, 1032 and 1033, 1025 and
1036, 1038 and 1039, 1041 and 1042, 1044 and 1045, 1047 and 1048,
1050 and 1051, 1053 and 1054, 1056 and 1057, 1059 and 1060, 1062
and 1063, 1065 and 1066, 1068 and 1069, 1071 and 1072, 1074 and
1075, 1077 and 1078, 1080 and 1081, 1083 and 1084, 1086 and 1087,
1089 and 1090, 1092 and 1093, 1095 and 1096, 1098 and 1099, 1101
and 1102, 1104 and 1105, 1107 and 1108, 1110 and 1111, 1113 and
1114, 1116 and 1117, 1119 and 1120, 1122 and 1123, 1125 and 1126,
1128 and 1129, 1131 and 1132, 1134 and 1135, 1137 and 1138, 1140
and 1141, 1143 and 1144, 1146 and 1147, 1149 and 1150, 1152 and
1153, 1155 and 1156, 1158 and 1159, 1161 and 1162, 1164 and 1165,
1167 and 1168, 1170 and 1171, 1173 and 1174, 1176 and 1177, 1179
and 1180, 1182 and 1183, 1185 and 1186, 1188 and 1189, 1191 and
1192, 1194 and 1195, 1197 and 1198, 1200 and 1201, 1203 and 1204,
1206 and 1207, 1209 and 1210, 1212 and 1213, 1215 and 1216, 1218
and 1219, 1221 and 1222, 1224 and 1225, 1227 and 1228, 1230 and
1231, 1233 and 1234, 1236 and 1237, 1239 and 1240, 1242 and 1243,
1245 and 1246, 248 and 1249, 1251 and 1252, 1254 and 1255, 1257 and
1258, 1260 and 1261, 1263 and 1264, 1266 and 1267, 1269 and 1270,
1272 and 1273, 1275 and 1276, 1278 and 1279, 1281 and 1282, 1284
and 1285, 1287 and 1288, 1290 and 1291, 1293 and 1294, 1296 and
1297, 1299 and 1300, 1302 and 1303, 1305 and 1306, 1308 and 1309,
1311 and 1312, 1314 and 1315, 1317 and 1318, 1320 and 1321, 1323
and 1324, 1326 and 1327, 1329 and 1330, 1332 and 1333, 1335 and
1336, 1338 and 1339, 1341 and 1342, 1344 and 1345, 1347 and 1348,
1350 and 1351, 1353 and 1354, 1356 and 1357, 1359 and 1360, 1362
and 1363, 1365 and 1366, 1368 and 1369, 1371 and 1372, 1374 and
1375, 1377 and 1378, 1380 and 1381, 1383 and 1384, 1386 and 1387,
1389 and 1390, 1392 and 1393, 1395 and 1396, 1398 and 1399, 1401
and 1402, 1404 and 1405, 1407 and 1408, 1410 and 1411, 1413 and
1414, 1416 and 1417, 1419 and 1420, 1422 and 1423, 1425 and 1426,
1428 and 1429, 1431 and 1432, 1434 and 1435, 1437 and 1438, 1440
and 1441, 1443 and 1444, 1446 and 1447, 1449 and 1450, 1452 and
1453, 1455 and 1456, 1458 and 1459, 1461 and 1462, 1464 and 1465,
1467 and 1468, 1470 and 1471, 1473 and 1474, 1476 and 1477, 1479
and 1480, 1482 and 1483, 1485 and 1486, 1488 and 1489, 1491 and
1492, 1494 and 1495, 1497 and 1498, 1500 and 1501, 1503 and 1504,
1506 and 1507, 1509 and 1510, 1512 and 1513, 1515 and 1516, 1518
and 1519, 1521 and 1522, 1524 and 1525, 1527 and 1528, 1530 and
1531, 1533 and 1534, 1536 and 1537, 1539 and 1540, 1542 and 1543,
1545 and 1546, 1548 and 1549, 1551 and 1552, 1554 and 1555, 1557
and 1558, 1560 and 1561, 1563 and 1564, 1566 and 1567, 1569 and
1570, 1572 and 1573, 1575 and 1576, 1578 and 1579, 1581 and 1582,
1584 and 15685, 1587 and 1588, 1590 and 1591, 1593 and 1594, 1596
and 1597, 1599 and 1600, 1602 and 1603, 1605 and 1606, 1608 and
1609, 1611 and 1612, 1614 and 1615, 1617 and 1618, 1620 and 1621,
1623 and 1624, 1626 and 1627, 1629 and 1630, 1632 and 1633, 1635
and 1636, 1638 and 1639, 1641 and 1642, 1644 and 1645, 1647 and
1648, 1650 and 1651, 1653 and 1654, 1656 and 1657, 1659 and 1660,
1662 and 1663, 1665 and 1666, 1668 and 1669, 1671 and 1672, 1674
and 1675, 1677 and 1678, 1680 and 1681, 1683 and 1684, 1686 and
1687, 1689 and 1690, 1692 and 1693, 1695 and 1696, 1698 and 1699,
1701 and 1702, 1704 and 1705, 1707 and 1708, 1710 and 1711, 1713
and 1714, 1716 and 1717, 1719 and 1720, 1722 and 1723, 1725 and
1726, 1728 and 1729, 1731 and 1732, 1734 and 1735, 1737 and 1738,
1740 and 1741, 1743 and 1744, 1746 and 1747, 1749 and 1750, 1752
and 1753, 1755 and 1756, 1758 and 1759, 1761 and 1762, 1764 and
1765, 1767 and 1768, 1770 and 1771, 1773 and 1774, 1776 and 1777,
1779 and 1780, 1782 and 1783, 1785 and 1786, 1788 and 1789, 1791
and 1792, 1794 and 1795, 1797 and 1798, 1800 and 1801, 1803 and
1804, 1806 and 1807, 1809 and 1810, 1812 and 1813, 1815 and 1816,
1818 and 1819, 1821 and 1822, 1824 and 1825, 1827 and 1828, 1830
and 1831, 1833 and 1834, 1836 and 1837, 1839 and 1840, 1842 and
1843, 1845 and 1846, 1848 and 1849, 1851 and 1852, 1854 and 1855,
1857 and 1858, 1860 and 1861, 1863 and 1864, 1866 and 1867, 1869
and 1870, 1872 and 1873, 1875 and 1876, 1878 and 1879, 1881 and
1882, 1884 and 1885, 1887 and 1888, 1890 and 1891, 1893 and 1894,
1896 and 1897, 1899 and 1900, 1902 and 1903, 1905 and 1906, 1908
and 1909, 1911 and 1912, 1914 and 1915, 1917 and 1918, 1920 and
1921, 1923 and 1924, 1926 and 1927, 1929 and 1930, 1932 and 1933,
1925 and 1936, 1938 and 1939, 1941 and 1942, 1944 and 1945, 1947
and 1948, 1950 and 1951, 1953 and 1954, 1956 and 157, 1959 and
1960, 1962 and 1963, 1965 and 1966, 1968 and 1969, 1971 and 1972,
1974 and 175, 1977 and 1978, 1980 and 1981, 1983 and 1984, 1986 and
1987, 1989 and 1999, 1992 and 1993, 1995 and 1996, 1998 and 1999,
2001 and 2002, 2004 and 2005, 2007 and 2008, 2010 and 2011, 2013
and 2014, 2016 and 2017, 2019 and 2020, 2022 and 2023, 2025 and
2026, 2028 and 2029, 2031 and 2032, 2034 and 2035, 2037 and 2038,
2040 and 2041, 2043 and 2044, 2046 and 2047, 2049 and 2050, 2052
and 2053, 2055 and 2056, or 2058 and 2059 of an NS3/4A nucleic acid
sequence such as SEQ ID NO: 1, or between any of the codons of a
nucleic acid sequence encoding an NS3/4A variant (e.g., SEQ ID NO:
35). For example, in some embodiments, a nucleic acid encoding a
TCE or a TCE and a linker(s) and/or an adjuvant sequence is
inserted between nucleotides 1370 and 1548 of SEQ ID NO: 35, or in
an analogous position in any NS3/4A nucleic acid. Embodiments also
relate to polypeptides encoded by said nucleic acids.
[0236] In some embodiments, the nucleic acid sequences encoding the
TCE or TCE and linker and/or adjuvant sequence portion of the
chimeric NS3/4A polypeptide can be juxtaposed to the 5' end of the
NS3/4A sequences, and encoding a chimeric NS3/4A polypeptide with a
TCE or TCE and linker(s) and/or adjuvant sequence on the N-terminal
end of the NS3/4A polypeptide. In some embodiments, the nucleic
acid sequences encoding the TCE or TCE and linker(s) and/or
adjuvant sequence polypeptide can be flanking (e.g., juxtaposed to)
the 3' end of the NS3/4A sequences and encode a chimeric NS3/4A
polypeptides with a TCE or TCE and linker(s) and/or adjuvant
sequence on the C-terminal end of the NS3/4A polypeptide. In
embodiments in which the chimeric NS3/4A polypeptide comprises more
than one TCE or TCE and linker(s) and/or adjuvant sequence, the
nucleic acids encoding the TCEs can be located different positions
relative to the nucleic acids encoding the NS3/4A sequences (i.e.,
5', within, or 3') and relative to each other. Optionally, NS3/4A
variants include a substitution of at least one amino acid with any
other amino acid in one or more of the domains of a different
NS3/NS4A sequences. Embodiments also relate to polypeptides encoded
by said nucleic acid sequences.
[0237] The skilled artisan will readily appreciate that a variety
of techniques can be used to generate variants, such as the
generation of insertions of desired sequences (e.g., TCEs and
linkers) within NS3/4A nucleic acid and polypeptide sequences
described herein. For example, overlapping PCR can be used to
generate desired substitutions or insertions (e.g., a nucleic acid
encoding a TCE, and/or linker sequences) within the NS3/4A
sequences, or at the 3' or 5' ends of the NS3/4A sequences. (See,
e.g., Ho et al. (1989), Gene 77(1):51-9). Several commercially
available kits are also available to facilitate site-directed
mutagenesis, to facilitate the generation of NS3/4A variants, such
as the recombinant nucleic acids and encoded polypeptides disclosed
herein. An exemplary commercially available kit useful for
generating chimeric NS3/4A polypeptides and chimeric NS3/4A
polypeptide variants is the QUICKCHANGE.RTM. site directed
mutagenesis kit (Stratagene, La Jolla, Calif.).
[0238] In preferred embodiments, the catalytic activity (e.g., the
protease or helicase activity) of a chimeric NS3/4A or chimeric
NS3/4A variant may be enhanced or unchanged, relative to the native
polypeptide, or may be diminished by less than 50%, and preferably
less than 20% relative to the native polypeptide. In some
embodiments the protease activity of an NS3/4A chimeric polypeptide
or chimeric polypeptide variant is diminished by less than 30%,
25%, 20%, 19%, 18%, 17%, 16%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%,
2%, 1%, or 0.5%, relative to the native polypeptide. In some
embodiments the protease activity of an NS3/4A variant may be
enhanced by at least 30%, 25%, 20%, 19%, 18%, 17%, 16%, 15%, 10%,
9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.5%, relative to the native
polypeptide. Exemplary NS3/4A variants with altered protease
activity are discussed in further detail herein.
[0239] In preferred embodiments, the NS3/4A chimeric polypeptide
retains protease activity. Accordingly, in some embodiments, the
nucleic acids encoding the chimeric NS3/4A polypeptide encode or
the chimeric NS3/4A polypeptides comprise the native amino acid
sequence at the following positions of the NS3/4A sequence: Leu44,
Ile48, Trp53, His57, Asp81, Trp85, Ala91, Leu94, Cys97, Cys99,
Leu106, Thr108, Arg123, Gly124, Leu126, Ser139, Gly140, Leu143,
Leu144, Cys145, His149, Ile153, Phe169, and Leu175. That is, the
aforementioned residues are unchanged in some embodiments or, in
some embodiments, the nucleic acids encoding TCEs or TCEs and
linkers and/or adjuvant sequence are not substituted for, inserted
within, or inserted at positions adjacent to nucleic acid sequences
encoding the following amino acids of NS3/4A sequences: Leu44,
Ile48, Trp53, His57, Asp81, Trp85, Ala91, Leu94, Cys97, Cys99,
Leu106, Thr108, Arg123, Gly124, Leu126, Ser139, Gly140, Leu143,
Leu144, Cys145, His149, Ile153, Phe169, and Leu175.
[0240] In some embodiments, the chimeric NS3/4A variants exhibit
enhanced protease activity. Embodiments disclosed herein provide
NS3/4A chimeric polypeptides including one or more of the following
amino acid substitutions in the NS3/4A sequence: Tyr6Ala, Arg11Ala,
Leu13Ala, Leu14Ala, Glu30Ala, Cys52Ala, Gly58Ala, Ala59Gly,
Ile64Ala, Ile64Ala, Gln73Ala, Thr76Ala, Pro86Ala, Ala111Gly, Gly
122Ala, Tyr 134Ala, Lys 136Ala, Gly 141Ala, Val158Ala, Arg161Ala,
Ala166Gly, or Thr177Ala. That is, in some embodiments one or more
of the following amino acid substitutions are included: Tyr6Ala,
Arg11Ala, Leu13Ala, Leu14Ala, Glu30Ala, Cys52Ala, Gly58Ala,
Ala59Gly, Ile64Ala, Ile64Ala, Gln73Ala, Thr76Ala, Pro86Ala,
Ala111Gly, Gly 122Ala, Tyr 134Ala, Lys 136Ala, Gly 141Ala,
Val158Ala, Arg161Ala, Ala166Gly, or Thr177Ala.
[0241] In some embodiments, NS3/4A variant sequences used in the
embodiments disclosed herein lack a proteolytic cleavage site, such
as SEQ. ID. NOs.: 14 and 16-26. In some embodiments, fragments of
the NS3/4A variant sequences containing any number of consecutive
amino acids between at least 3-300 amino acids (e.g., 3, 4, 6, 8,
10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, or
300 consecutive amino acids) of any one of SEQ. ID. NOs.: 14 and
16-26 are used in the embodiments disclosed herein. Other exemplary
NS3/4A variants with altered protease activity may generally be
identified by modifying one or more of the above nucleic acid or
polypeptide sequences and evaluating the protease activity of the
variant, as discussed in further detail in Example 1.
[0242] In some embodiments, the helicase activity of a NS3/4A
variant is diminished by less than 30%, 25%, 20%, 19%, 18%, 17%,
16%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.5%,
relative to the native polypeptide. In some embodiments the
helicase activity of an NS3/4A variant may be enhanced by at least
30%, 25%, 20%, 19%, 18%, 17%, 16%, 15%, 10%, 9%, 8%, 7%, 6%, 5%,
4%, 3%, 2%, 1%, or 0.5%, relative to the native polypeptide. Such
variants may generally be identified by modifying one of the above
polypeptide sequences and evaluating the helicase activity as
described herein
[0243] Guidance in determining the identity of amino acids that may
affect NS3 helicase activity can be found by comparing the sequence
of the NS3 helicase domain with that of homologous known protein
molecules and minimizing the number of amino acid sequence changes
made in regions of high homology. An exemplary assay for testing
variants for helicase activity is discussed in Artsaenko O, et al.,
(2003) J Gen Virol. 2003 84(Pt 9):2323-32, and Zhang et al., (2005)
J Virol. 79(14):8687-97; and Kyono et al., (2004) J Biochem (Tokyo)
135(2):245-52.
[0244] Optionally, chimeric NS3/4A variants can encode or comprise
amino acid substitutions, wherein one amino acid is substituted
with another amino acid having similar structural and/or chemical
properties (e.g., conservative amino acid replacements). A list of
conservative amino acid substitutions can be found in Table 1.
TABLE-US-00002 TABLE 1 Original Exemplary Preferred Residue
Substitutions Substitutions Ala (A) val; leu; ile val Arg (R) lys;
gln; asn lys Asn (N) gln; his; lys; arg gln Asp (D) glu glu Cys (C)
ser ser Gln (Q) asn asn Glu (E) asp asp Gly (G) pro; ala ala His
(H) asn; gln; lys; arg arg Ile (I) leu; val; met; ala; phe; leu
norleucine Leu (L) norleucine; ile; val; ile met; ala; phe Lys (K)
arg; gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile;
ala; tyr leu Pro (P) ala ala Ser (S) thr thr Thr (T) ser ser Trp
(W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile; leu;
met; phe; leu ala; norleucine
[0245] In some embodiments, variant NS3/4A sequences are engineered
or optimized for codons most frequently used in humans. The nucleic
acid sequence of an exemplary codon-optimized NS3/4A nucleic acid
sequence (coNS3/4A) is provided in SEQ. ID. NO.: 35. The peptide
encoded by said nucleic acid sequence is provided in SEQ. ID. NO.:
36. The skilled artisan will appreciate, however, that any HCV
NS3/4A sequences disclosed herein or discovered in the future can
be used to generate codon-optimized variants and that all
codon-optimized variants are within the scope of the present
invention.
[0246] The nucleic acid and corresponding NS3/4A peptide (SEQ ID
NOs: 35 and 36) do not correspond to any known HCV sequence or
genome. The codon-optimized NS3/4A encoding nucleic acid was found
to be only 79% homologous, within the region of nucleotide
positions 3417-5475, to HCV-1 and contained a total of 433
different nucleotides. The NS3/4A peptide encoded by the
codon-optimized nucleic acid sequence is only 98% homologous to
HCV-1 and contained a total of 15 different amino acids. As
demonstrated in Example 2, below, the codon optimized nucleic acid
was found to generate a higher expression level of NS3 and was
found to be more immunogenic, with respect to both humoral and
cellular responses, as compared to the native NS3/4A gene from
which it was derived. Accordingly, in preferred embodiments, the
NS3/4A nucleic acid sequences encoding, or the encoded polypeptide
sequences of the NS3/4A chimeric polypeptides comprise
codon-optimized nucleic acid and polypeptide sequences of native
HCV sequences. For example, in some embodiments, the NS3/4A nucleic
acid sequences or encoded polypeptide sequences of an NS3/4A
chimeric polypeptides comprises SEQ ID NO: 35 or SEQ ID NO: 36, or
fragments thereof, or variants thereof and TCEs and/or linker
sequences.
Example 2
[0247] The sequence of the unique NS3/4A gene described in Example
1 (SEQ. ID. NO.: 1) was analyzed for codon usage with respect to
the most commonly used codons in human cells. A total of 435
nucleotides were replaced to optimize codon usage for human cells.
The sequence was sent to Retrogen Inc. (6645 Nancy Ridge Drive, San
Diego, Calif. 92121) and they were provided with instructions to
generate a full-length synthetic codon optimized NS3/4A gene. The
codon optimized NS3/4A gene had a sequence homology of 79% within
the region between nucleotide positions 3417-5475 of the HCV-1
reference strain. A total of 433 nucleotides differed. On an amino
acid level, the homology with the HCV-1 strain was 98% and a total
of 15 amino acids differed.
[0248] The full length codon optimized 2.1 kb DNA fragment of the
HCV corresponding to the amino acids 1007 to 1711 encompassing the
NS3 and NS4A NS3/4A gene fragment was amplified by the polymerase
chain reaction (PCR) using high fidelity polymerase (Expand High
Fidelity PCR, Boehringer-Mannheim, Mannheim, Germany). The amplicon
was then inserted into a Bam HI and XbaI digested pVAX vector
(Invitrogen, San Diego), which generated the MSLF1-pVAX
(coNS3/4A-pVAX) plasmid. All expression constructs were sequenced.
Plasmids were grown in competent BL21 E. Coli. The plasmid DNA used
for in vivo injection was purified using Qiagen DNA purification
columns, according to the manufacturers instructions (Qiagen GmbH,
Hilden, FRG). The concentration of the resulting plasmid DNA was
determined spectrophotometrically (Dynaquant, Pharmacia Biotech,
Uppsala, Sweden) and the purified DNA was dissolved in sterile
phosphate buffer saline (PBS) at concentrations of 1 mg/ml.
[0249] The expression of NS3 and NS3/4A proteins from the wtNS3/4A
(wild-type NS3/4A) and coNS3/4A plasmids, were analyzed by an in
vitro transcription and translation assay. The assay showed that
the proteins could be correctly translated from the plasmids and
that the coNS3/4A plasmid gave detectable NS3 and NS3/4A bands at a
higher plasmid dilution as compared to the wtNS3/4A plasmid. This
result provided strong evidence that the in vitro translation from
the coNS3/4A plasmid is more effective than wtNS3/4A. To compare
the expression levels more precisely, HepG2 cells were transiently
transfected with the wtNS3/4A and the coNS3/4A plasmids. These
experiments revealed that the coNS3/4A plasmid generated 11-fold
higher expression levels of the NS3 protein when compared to the
wtNS3/4A plasmid, as determined by densitometry and a standard
curve of recombinant NS3. Since the wtNS3/4A and the coNS3/4A
plasmids are identical in size it is unlikely that there would be
any major differences in transfections efficiencies between the
plasmids. Staining of coNS3/4A plasmid transfected, and SFV
infected, BHK cells revealed a similar perinuclear and cytoplasmic
distribution of the NS3 as previously observed, confirming an
unchanged subcellular localization.
[0250] TABLE 2 describes the sequence of the proteolytic cleavage
site of NS3/4A, referred to as the breakpoint between NS3 and NS4A.
This wild-type breakpoint sequence was mutated in many different
ways so as to generate several different NS3/4A breakpoint mutants.
TABLE 2 also identifies these mutant breakpoint sequences. The
fragments listed in TABLE 2 are preferred immunogens that can be
incorporated with or without an adjuvant (e.g., ribavirin) into a
composition for administration to an animal so as to induce an
immune response in said animal to HCV.
TABLE-US-00003 TABLE 2 Deduced amino SEQ Plasmid acid sequence ID
NS3/4A-pVAX TKYMTCMSADLEVVTSTWVLVGGVL 14 NS3/4A-TGT-pVAX
TKYMTCMSADLEVVTGTWVLVGGVL 16 NS3/4A-RGT-pVAX
TKYMTCMSADLEVVRGTWVLVGGVL 17 NS3/4A-TPT-pVAX
TKYMTCMSADLEVVTPTWVLVGGVL 18 NS3/4A-RPT-pVAX
TKYMTCMSADLEVVRPTWVLVGGVL 19 NS3/4A-RPA-pVAX
TKYMTCMSADLEVVRPAWVLVGGVL 20 NS3/4A-CST-pVAX
TKYMTCMSADLEVVCSTWVLVGGVL 21 NS3/4A-CCST-pVAX
TKYMTCMSADLEVCCSTWVLVGGVL 22 NS3/4A-SSST-pVAX
TKYMTCMSADLEVSSSTWVLVGGVL 23 NS3/4A-SSSSCST-pVAX
TKYMTCMSADSSSSCSTWVLVGGVL 24 NS3A/4A-VVVVTST-pVAX
TKYMTCMSADVVVVTSTWVLVGGVL 25 NS5-pVAX ASEDVVCCSMSYTWTG 27
NS5A/B-pVAX SSEDVVCCSMWVLVGGVL 26 *The wild type sequence for the
NS3/4A fragment is NS3/4A-pVAX. The NS3/4A breakpoint is identified
by underline, wherein the P1 position corresponds to the first Thr
(T) and the P1' position corresponds to the next following amino
acid the NS3/4A-pVAX sequence. In the wild type NS3/4A sequence the
NS3 protease cleaves between the P1 and P1' positions.
[0251] To change the proteolytic cleavage site between NS3 and
NS4A, the NS3/4A-pVAX plasmid was mutagenized using the
QUICKCHANGE.TM. mutagenesis kit (Stratagene), following the
manufacturer's recommendations. To generate the "TPT" mutation, for
example, the plasmid was amplified using the primers
5'-CTGGAGGTCGTCACGCCTACCTGGGTGCTCGTT-3' (SEQ. ID. NO.: 31) and
5'-ACCGAGCACCCAGGTAGGCGTGACGACCTCCAG-3' (SEQ. ID. NO.: 32)
resulting in NS3/4A-TPT-pVAX. To generate the "RGT" mutation, for
example, the plasmid was amplified using the primers
5'-CTGGAGGTCGTCCGCGGTACCTGGGTGCTCGTT-3' (SEQ. ID. NO.: 33) and
5'-ACCGAGCACCCAGGTACC-GCGGACGACCTCCAG-3' (SEQ. ID. NO.: 34)
resulting in NS3/4A-RGT-pVAX. All mutagenized constructs were
sequenced to verify that the mutations had been correctly made.
Plasmids were grown in competent BL21 E. coli.
[0252] On an amino acid level, the homology with the HCV-1 strain
was 98% and a total of 15 amino acids differed. The nucleic acid
sequence of the codon-optimized NS3/4a is provided in SEQ ID NO:
35, whereas the peptide encoded by said nucleic acid sequence is
provided in SEQ ID NO: 36. The full length codon optimized 2.1 kb
DNA fragment of the HCV corresponding to the amino acids 1007 to
1711 encompassing the NS3 and NS4A NS3/4A gene fragment was
amplified by the polymerase chain reaction (PCR) using high
fidelity polymerase (Expand High Fidelity PCR, Boehringer-Mannheim,
Mannheim, Germany). The amplicon was then inserted into a Bam HI
and XbaI digested pVAX vector (Invitrogen, San Diego), which
generated the MSLF1-pVAX (coNS3/4A-pVAX) plasmid. All expression
constructs were sequenced. Plasmids were grown in competent BL21 E.
Coli. The plasmid DNA used for in vivo injection was purified using
Qiagen DNA purification columns, according to the manufacturers
instructions (Qiagen GmbH, Hilden, FRG). The concentration of the
resulting plasmid DNA was determined spectrophotometrically
(Dynaquant, Pharmacia Biotech, Uppsala, Sweden) and the purified
DNA was dissolved in sterile phosphate buffer saline (PBS) at
concentrations of 1 mg/ml.
[0253] The expression of NS3 and NS3/4A proteins from the wtNS3/4A
(wild-type NS3/4A) and coNS3/4A plasmids, were analyzed by an in
vitro transcription and translation assay. The assay showed that
the proteins could be correctly translated from the plasmids and
that the coNS3/4A plasmid gave detectable NS3 and NS3/4A bands at a
higher plasmid dilution as compared to the wtNS3/4A plasmid. This
result provided strong evidence that the in vitro translation from
the coNS3/4A plasmid is more effective than wtNS3/4A. To compare
the expression levels more precisely, HepG2 cells were transiently
transfected with the wtNS3/4A and the coNS3/4A plasmids. These
experiments revealed that the coNS3/4A plasmid generated 11-fold
higher expression levels of the NS3 protein when compared to the
wtNS3/4A plasmid, as determined by densitometry and a standard
curve of recombinant NS3. Since the wtNS3/4A and the coNS3/4A
plasmids are identical in size it is unlikely that there would be
any major differences in transfections efficiencies between the
plasmids. Staining of coNS3/4A plasmid transfected, and SFV
infected, BHK cells revealed a similar perinuclear and cytoplasmic
distribution of the NS3 as previously observed, confirming an
unchanged subcellular localization.
[0254] Several nucleic acid embodiments include nucleotides
encoding the HCV peptides described herein (SEQ. ID. NOs.: 2-11 or
SEQ. ID. NO.: 36) or a fragment thereof (e.g., SEQ. ID. NOs.: 14
and 15) containing any number of consecutive amino acids between at
least 3-50 (e.g., 3, 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45,
or 50 amino acids in length). Some embodiments for example, include
genomic DNA, RNA, and cDNA encoding these HCV peptides. The HCV
nucleotide embodiments not only include the DNA sequences shown in
the sequence listing (e.g., SEQ. ID. NO.: 1 or SEQ. ID. NO.: 35)
but also include nucleotide sequences encoding the amino acid
sequences shown in the sequence listing (e.g., SEQ. ID. NOs.: 2-11
or SEQ. ID. NO.: 36) and any nucleotide sequence that hybridizes to
the DNA sequences shown in the sequence listing under stringent
conditions (e.g., hybridization to filter-bound DNA in 0.5 M
NaHPO.sub.4, 7.0% sodium dodecyl sulfate (SDS), 1 mM EDTA at
50.degree. C.) and washing in 0.2.times.SSC/0.2% SDS at 50.degree.
C. and any nucleotide sequence that hybridizes to the DNA sequences
that encode an amino acid sequence provided in the sequence listing
(SEQ. ID. NOs.: 2-11 or SEQ. ID. NO.: 36) under less stringent
conditions (e.g., hybridization in 0.5 M NaHPO.sub.4, 7.0% sodium
dodecyl sulfate (SDS), 1 mM EDTA at 37.degree. C. and washing in
0.2.times.SSC/0.2% SDS at 37.degree. C.).
[0255] The nucleic acid embodiments of the invention also include
fragments, modifications, derivatives, and variants of the
sequences described above. Desired embodiments, for example,
include nucleic acids having at least 25 consecutive bases of one
of the novel HCV sequences or a sequence complementary thereto and
preferred fragments include at least 25 consecutive bases of a
nucleic acid encoding the NS3/4A molecule of SEQ. ID. NO.: 2 or
SEQ. ID. NO.: 36 or a mutant NS3/4A molecule of SEQ. ID. NOs.: 3-13
or a sequence complementary thereto.
[0256] In this regard, the nucleic acid embodiments described
herein can have any number of consecutive nucleotides between about
12 to approximately 2112 consecutive nucleotides of SEQ. ID. NO.: 1
or SEQ. ID. NO.: 35. Some DNA fragments, for example, include
nucleic acids having at least 12-15, 15-20, 20-30, 30-50, 50-100,
100-200, 200-500, 500-1000, 1000-1500, 1500-2079, or 1500-2112
consecutive nucleotides of SEQ. ID. NO.: 1 or SEQ. ID. NO.: 35 or a
complement thereof. These nucleic acid embodiments can also be
altered by substitution, addition, or deletion so long as the
alteration does not significantly affect the structure or function
(e.g., ability to serve as an immunogen) of the HCV nucleic acid.
Due to the degeneracy of nucleotide coding sequences, for example,
other DNA sequences that encode substantially the same HCV amino
acid sequence as depicted in SEQ. ID. NOs.: 2-13 or SEQ. ID. NO.:
36 can be used in some embodiments. These include, but are not
limited to, nucleic acid sequences encoding all or portions of HCV
peptides (SEQ. ID. NOs.: 2-13) or nucleic acids that complement all
or part of this sequence that have been altered by the substitution
of different codons that encode a functionally equivalent amino
acid residue within the sequence, thus producing a silent change,
or a functionally non-equivalent amino acid residue within the
sequence, thus producing a detectable change. Accordingly, the
nucleic acid embodiments of the invention are said to be
comprising, consisting of, or consisting essentially of nucleic
acids encoding any one of SEQ. ID. NOs.: 2-27 or SEQ. ID. NO.: 36
in light of the modifications above.
[0257] In some embodiments, the HCV NS3/4A sequences comprise,
consist essentially of, or consist of NS3 and or NS4A sequences
that encode fragments, or functional fragments of the full-length
NS3/4A polypeptide. Such fragments may be truncated at the
N-terminus or C-terminus, or may lack internal residues, for
example, when compared with a full length native protein. In
preferred embodiments, NS3/4A fragments lack amino acid residues
that are not essential for the catalytic activity of the NS3
polypeptide. For example, in some embodiments the NS3/4A sequences
can comprise, consist, or consist essentially of fragments of any
of SEQ ID NOs: 1, 35, and 572-808 encoding any number of
consecutive amino acids (e.g., 3, 4, 6, 8, 10, 12, 15, 20, 25, 30,
35, 40, 45, 50, 75, 100, 150, 200, 250, or 300 consecutive amino
acids). The section below describes several antigens that can be
used in the compositions and methods described herein.
[0258] Epitopes
[0259] The chimeric NS3/4A nucleic acids and polypeptides disclosed
herein include sequences comprising antigens, such as TCEs,
positioned at non-naturally occurring locations, either within
and/or flanking (e.g., juxtaposed to) NS3/4A sequences. As used
herein, the term epitope refers to a set of amino acid residues
that are involved in recognition by a particular immunoglobulin
(i.e., a B cell epitope or "BCE") or in the context of TCE, those
residues necessary for recognition by T cell receptor proteins
and/or Major Histocompatability Complex (MHC) receptors. In an
immune system setting, in vivo or in vitro, an epitope is the
collective features of a molecule, such as primary, secondary, and
tertiary peptide structure, and charge, that together form a site
recognized by an immunoglobulin, T cell receptor, or HLA molecule.
TCEs are recognized by either CD4+ T cells, or helper T lymphocytes
("HTLs") or CD8+ T cells, or cytotoxic T lymphocytes ("CTLs").
[0260] TCEs generally comprise a chain of at least four amino acid
residues, preferably at least six, more preferably eight to ten,
sometimes eleven to fourteen residues, and usually fewer than about
thirty residues, more usually fewer than about twenty-five, and
preferably fewer than fifteen, e.g., eight to fourteen amino acid
residues derived from selected epitopic regions of the target
antigen(s). It is to be appreciated, however, that TCE nucleic
acids, or TCE amino acid sequences can refer to nucleic acids
encoding or protein or peptide molecules, larger than and
comprising an epitope of the invention are still within the scope
of the invention. For example, nucleic acid and polypeptide
sequences of full length proteins that contain at least one TCE,
that are capable of producing an immune response are contemplated
for use in some embodiments.
[0261] In some aspects, the TCE are nucleic acids encoding peptides
wherein said nucleic acids are, are at least, are at least about,
are less than, or are less than about 3 nucleotides, 4 nucleotides,
5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9
nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13
nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17
nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21
nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25
nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29
nucleotides, 30 nucleotides, 31 nucleotides, 32 nucleotides, 33
nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleotides, 37
nucleotides, 38 nucleotides, 39 nucleotides, 40 nucleotides, 41
nucleotides, 42 nucleotides, 43 nucleotides, 44 nucleotides, 45
nucleotides, 46 nucleotides, 47 nucleotides, 48 nucleotides, 49
nucleotides, 50 nucleotides, 55 nucleotides, 60 nucleotides, 65
nucleotides, 70 nucleotides, 75 nucleotides, 80 nucleotides, 85
nucleotides, 90 nucleotides, 95 nucleotides, 100 nucleotides, 110
nucleotides, 120 nucleotides, 130 nucleotides, 140 nucleotides, 150
nucleotides, 160 nucleotides, 170 nucleotides, 180 nucleotides, 190
nucleotides, 200 nucleotides, 250 nucleotides, 300 nucleotides, 350
nucleotides, 400 nucleotides, 450 nucleotides, 500 nucleotides, 550
nucleotides, 600 nucleotides, 650 nucleotides, 700 nucleotides, 750
nucleotides, 800 nucleotides, 850 nucleotides, 900 nucleotides, 950
nucleotides, 1000 nucleotides, 1100 nucleotides, 1200 nucleotides,
1300 nucleotides, 1400 nucleotides, 1500 nucleotides, 1600
nucleotides, 1700 nucleotides, 1800 nucleotides, 1900 nucleotides,
2000 nucleotides, 2500 nucleotides, 3000 nucleotides, 3500
nucleotides, 4000 nucleotides, 4500 nucleotides, 5000 nucleotides,
6000 nucleotides, 7000 nucleotides, 8000 nucleotides, 9000
nucleotides, 10,000 nucleotides in length
[0262] In some aspects, the TCE are peptides or peptide fragments
that are, are at least, are at least about, are less than, or are
less than about 3 amino acids, 4 amino acids, 5 amino acids, 6
amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino
acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino
acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino
acids, 19 amino acids, 20 amino acids, 21 amino acids, 22 amino
acids, 23 amino acids, 24 amino acids, 25 amino acids, 26 amino
acids, 27 amino acids, 28 amino acids, 29 amino acids, 30 amino
acids, 31 amino acids, 32 amino acids, 33 amino acids, 34 amino
acids, 35 amino acids, 36 amino acids, 37 amino acids, 38 amino
acids, 39 amino acids, 40 amino acids, 41 amino acids, 42 amino
acids, 43 amino acids, 44 amino acids, 45 amino acids, 46 amino
acids, 47 amino acids, 48 amino acids, 49 amino acids, 50 amino
acids, 55 amino acids, 60 amino acids, 65 amino acids, 70 amino
acids, 75 amino acids, 80 amino acids, 85 amino acids, 90 amino
acids, 95 amino acids, 100 amino acids, 110 amino acids, 120 amino
acids, 130 amino acids, 140 amino acids, 150 amino acids, 160 amino
acids, 170 amino acids, 180 amino acids, 190 amino acids, 200 amino
acids, 250 amino acids, 300 amino acids, 350 amino acids, 400 amino
acids, 450 amino acids, 500 amino acids, 550 amino acids, 600 amino
acids, 650 amino acids, 700 amino acids, 750 amino acids, 800 amino
acids, 850 amino acids, 900 amino acids, 950 amino acids, 1000
amino acids, 1100 amino acids, 1200 amino acids, 1300 amino acids,
1400 amino acids, 1500 amino acids, 1600 amino acids, 1700 amino
acids, 1800 amino acids, 1900 amino acids, 2000 amino acids, 2500
amino acids, 3000 amino acids, 3500 amino acids, 4000 amino acids,
4500 amino acids, 5000 amino acids, 6000 amino acids, 7000 amino
acids, 8000 amino acids, 9000 amino acids, 10,000 amino acids in
length.
[0263] Further, it will be appreciated that the term "TCE" includes
sequences that comprise one, two, or multiple TCEs. For example, a
TCE may refer to a recombinant string of CTL and/or HTL epitopes.
In some embodiments, the NS3/4A chimeric molecules disclosed herein
include epitope strings to generate a CTL response against any
chosen antigen/target that contains such epitopes. Optionally, HTL
epitopes which are active in individuals of different HLA types,
for example HTLs from tetanus (against which most individuals will
already be primed) are present in the embodiments disclosed herein.
Further, in some embodiments, in addition to a TCE, it may also be
useful to include B cell epitopes for stimulating B cell responses
and antibody production. Optionally, multiple epitope (e.g.
multiple TCE and/or multiple TCE and BCE) conjugates can be
engineered to be linked by a linker molecule. Linkers can comprise
relatively neutral amino acid sequences or amino acid mimetics,
such as, e.g., Ala, Gly, or other neutral linkers of nonpolar amino
acids or neutral polar amino acids. In certain preferred
embodiments herein the neutral linker is Ala. It will be understood
that the optionally present linker need not be comprised of the
same residues and thus may be a hetero- or homo-oligomer. Preferred
exemplary linkers are homo-oligomers of Ala or Gly. When present,
the linker will usually be at least one or two residues, more
usually three to six residues. Adjuvant sequences such as nucleic
acids encoding HIV TAT or fragments thereof (e.g., 3, 6, 9, 12, 15,
21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, or 60
nucleotides in length) can be employed in some embodiments.
Exemplary sequences can be found in WO05039631A1, which designates
the United States and was published in English, hereby expressly
incorporated by reference in its entirety. Optionally, linkers
and/or adjuvant sequences flank, or are juxtaposed to TCE sequences
and/or NS3/4A sequences.
[0264] The compositions and methods disclosed herein relate to
antigens against which it is desired to generate an immune
response. For example, the compositions and methods disclosed
herein are useful, for example, in generating or enhancing the
immunogenicity of TCEs derived from agents against which CD8.sup.+
T cell responses have been shown to play a protective role. As
such, the compositions disclosed herein are useful in diseases that
include but are not limited to infection and disease caused by the
viruses including, but not limited to: influenza A and B viruses
(FLU-A, FLU-B), human immunodeficiency type I and II viruses
(HIV-I, HIV-II), Epstein-Barr virus (EBV), human T lymphotropic (or
T-cell leukemia) virus type I and type II (HTLV-I, HTLV-II), human
papillomaviruses types 1 to 18 (HPV-1 to HPV-18), rubella (RV),
varicella-zoster (VZV), hepatitis B (HBV), hepatitis C (HCV),
adenoviruses (AV), and herpes simplex virus (HSV), cytomegalovirus
(CMV), poliovirus, respiratory syncytial (RSV), rhinovirus, rabies,
mumps, rotavirus or measles viruses. Further, the compositions
disclosed herein are useful in diseases caused by the bacteria
Mycobacterium tuberculosis and Listeria sp.; and by the protozoan
parasites Plasmodium, Toxoplasma and Trypanosoma and the like.
[0265] In a like manner, the compositions and methods described
herein are applicable to tumor-associated proteins (e.g., related
to melanoma, breast cancer, colon cancer and the like), which could
be sources for CTL epitopes. Such tumor proteins and/or peptides,
include, but are not limited to, products of the MAGE-1, -2 and -3
genes, products of the c-ErbB2 (HER-2/neu) proto-oncogene, tumor
suppressor and regulatory genes which could be either mutated or
overexpressed such as p53, ras, myc, and RB1. Tissue specific
proteins to target CTL responses to tumors such as prostatic
specific antigen (PSA) and prostatic acid phosphatase (PAP) for
prostate cancer, and tyrosinase for melanoma. In addition viral
related proteins associated with cell transformation into tumor
cells such as EBNA-1, HPV E6 and E7 are likewise applicable. A
large number of peptides from some of the above proteins have been
analyzed for the presence of MHC-binding motifs and for their
ability to bind with high efficiency to purified MHC molecules and
are the subject of pending patent applications (U.S. patent
application Ser. Nos. 08/159,339 and 08/073,205, previously
incorporated herein by reference)
[0266] The amino acid sequences of exemplary TCEs, including both
HTL and CTL TCEs, are listed in Table 3. Nucleic acids encoding
these sequences can be readily generated by those of skill in the
art.
TABLE-US-00004 TABLE 3 SEQ ID NO Epitope Source Sequence Source 222
EBV YLLEMLWRL US 6723695* 223 EBV YFLEILWGL US 6723695 224 EBV
YLLEILWRL US 6723695 225 EBV YLQQNWWTL US 6723695 226 EBV LLLALLFWL
US 6723695 227 EBV LLVDLLWLL US 6723695 228 EBV LLLIALWNL US
6723695 229 EBV WLLLFLAIL US 6723695 230 EBV TLLVDLLWL US 6723695
231 EBV LLWLLLFLA US 6723695 232 EBV ILLIIALYL US 6723695 233 EBV
VLFIFGCLL US 6723695 234 EBV RLGATIWQL US 6723695 235 EBV ILYFIAFAL
US 6723695 236 EBV SLVIVTTFV US 6723695 237 EBV LMIIPLINV US
6723695 238 EBV TLFIGSHVV US 6723695 239 EBV LIPETVPYI US 6723695
240 EBV QLTPHTKAV US 6723695 241 EBV QNGALAINTF US 6703024* 242 EBV
LLDFVRFMGV US 6703024 243 EBV QAKWRLQTL WO/95AU0000140* 244 EBV
RYSIFFDY WO/95AU0000140 245 EBV HLAAQGMAY WO/95AU0000140 246 EBV
YPLHEQHGM WO/95AU0000140 247 EBV SVRDRLARL WO/95AU0000140 248 EBV
AVLLHEESM WO/95AU0000140 249 EBV VSFIEFVGW, WO/95AU0000140 250 EBV
FRKAQIQGL WO/95AU0000140 251 EBV PYLFWLAAI, WO/95AU0000140 252 EBV
TVFYNIPPMPL WO/95AU0000140 253 EBV PGDQLPGFSDGRACPV WO/95AU0000140
254 EBV VEITPYKPTW WO/95AU0000140 255 EBV TYSAGIVQI US 6,699,477*
256 HPV AQIFNKPYW, US 6,911,207* 257 HPV AGVDNRECI US 6,911,207 258
MAGE-1 EADPTGHSY US 6,419,931* (melanoma) 259 MAGE-2 KMVELVHFL US
6419931 260 MAGE-2 KMVELVHFLL US 6419931 261 MAGE-3 LVFGIELMEV US
6419931 262 MAGE-1 KVLEYVIKV US 6419931 263 MAGE-1 KVADLVGFLL US
6419931 264 MAGE-3 KVAEFVHFL US 6419931 265 MAGE-1 CILESLFRA US
6419931 266 MAGE-1 FLWGPRALA US 6419931 267 MAGE-1 VMIAMEGGHA US
6419931 268 MAGE-1 LVLGTLEEV US 6419931 269 MAGE-1 ALREEEEGV US
6419931 270 MAGE-1 ALAETSYVKV US 6419931 271 MAGE-1 YVIKVSARV US
6419931 272 MAGE-1 RALAETSYV US 6419931 273 HIV nef84-94 AVDLSHFLK
US 6419931 274 EBV NA4 416-424 IVTDFSVIK US 6419931 275 HBc18-27
FLPSDFFPSV US 6419931 276 HIV RT ILKEPVHGV US 6419931 277 HTLV-1,
Tox 12-19 LFGYPVYV US 6419931 278 Influenza A, M1 GILGFVFTL US
6419931 58-66 279 HCMV, gB 619-628 IAGNSAYEYV US 6419931 280
Plasmodium GIEYLNKIQNSLSTEWSPCSVT US 6942866* falciparum 281
Plasmodium YLDKVRATVGTEWTPCSVT US 6942866 vivax 282 Plasmodium
EFVKQISSQLTEEWSQCSVT US 6942866 yoelli 283 p53 264-272 LLGRNSFEV US
6419931 284 HBVadr-ENV WLSLLVPFV US 6419931 (SAg335-343) 285
c-ErbB2 RFRELVSEFSRMARDPQ US 6419931 (HER-2/neu) (Breast cancer)
286 HIV nef73-82 QVPLRPMTYK US 6419931 287 HIV-1 NL43 env
RLRDLLLIVTR US 6419931 gp41768-778 288 HCV 141-151 STLPETTTVRR US
6419931 289 Influenza virus SRYWAIRTR US 6419931 NP 383-391 290 HIV
gag p24 KRWIILGLNK US 6419931 265-274 291 P. falciparum KPKDELDY US
6419931 circumsp. 368-375 292 P. falciparum KSKDLEDY US 6419931
circumsp. 368-375 293 P. falciparum KPNDKSLY US 6419931 liver Ag
1850-1857 294 HIV-2 TPYDINQML US 6419931 295 P. falciparum
KPIVQYDNF US 6419931 liverAg1786-1794 296 B53 self peptide
YPAEITLTW US 6419931 297 HIV gp41 586-593 YLKDQQLL US 6419931 298
Influenza virus ELRSRYWAI US 6419931 NP 380-388 299 EBV EBNA-3
FLRGRAYGI US 6419931 300 HIV gag261-269 GEIYKRWII US 6419931 301
HIV gag331-339 DCKTILKAL US 6419931 302 HIV pol185-193 DPKVKQWPL US
6419931 303 HIV gp41 586-593 YLKDQQLYL US 6419931 304 HIV gap p17.3
GGKKKYKLK US 6419931 305 HBV POL LLAQFTSAI US 6419931 306 HBV ENV
LLVPFVQWFV US 6419931 307 HBV ENV WLSLLVPFV US 6419931 308 HBV ENV
FLLAQFTSA US 6419931 309 HBV POL FLLSLGIHL US 6419931 310 HBV POL
ALMPLYACI US 6419931 311 HBV ENV ILLLCLIFLL US 6419931 312 HBV POL
KULYSHPI US 6419931 313 HBV ENV VLLDYQGML US 6419931 314 HBV ENV
LLPIFFCLWV US 6419931 315 HBV ENV VLQAGFFLL US 6419931 316 HBV POL
YLHTLWKAGI US 6419931 317 HBV POL YLHTLWKAGV US 6419931 318 HBV ENV
PLLPIFFCL US 6419931 319 HBV NUC ILSTLPETTV US 6419931 320 HCV NS4
LLFNILGGWV US 6419931 321 HCV CORE LLALLSCLTV US 6419931 322 HCV
NS4 YLVAYQATV US 6419931 323 HCV NS1/ENV FLLLADARV US 6419931 324
HCV NS4 ILAGYGAGV US 6419931 325 HCV CORE DLMGYIPLV US 6419931 326
HCV CORE YLLPRRGPRL US 6419931 327 NS1/ENV2 ALSTGLIHL US 6419931
328 HCV CORE LLALLSCLTI US 6419931 329 HCV NS5 RLIVFPDLGV US
6419931 330 HCV NS5 RLHGLSAFSL US 6419931 331 HCV NS4 ILGGWVAAQL US
6419931 332 HCV ENV1 SMVGNWAKV US 6419931 333 HCV NS3 YLVTRHADV US
6419931 334 HCV NS4 VLAALAAYCL US 6419931 335 HPV16 E7 LLMGTLGIV US
6419931 336 HPV16 E7 YMLDLQPET US 6419931
337 HPV16 E6 FAFRDLCIV US 6419931 338 HPV16 E7 TLGIVCPIC US 6419931
339 HPV16 E7 TLHEYMLDL US 6419931 340 HPV16 E7 GTLGIVCPI US 6419931
341 HPV16 E7 MLDLQPETT US 6419931 342 HPV16 E6 TIHDIILECV US
6419931 343 HIV VLAEAMSQV US 6419931 344 HIV LLWKGEGAVV US 6419931
345 HIV LLWKGEGAV US 6419931 346 HIV ILKEPVHGV US 6419931 347 HIV
IVGAETFYV US 6419931 348 HIV IIGAETFYV US 6419931 349 HIV LWVTVYYGV
US 6419931 350 HIV LMVTVYYGV US 6419931 351 HBc 11-27
ATVELLSFLPSDFFPSV US 6419931 352 HBc 91-110 Thr-Asn-Met-Gly-Leu- US
6322789* Lys-Phe-Arg-Gln-Leu- Leu-Trp-Phe-His-Ile-
Ser-Cys-Leu-Thr-Phe 353 HBenv 329-348 Ala-Ser-Ala-Arg-Phe- US
6322789 Ser-Trp-Leu-Ser-Leu- Leu-Val-Pro-Phe-Val-
Gln-Trp-Phe-Val-Gly 354 HBenv 349-368 Leu-Ser-Pro-Thr-Val- US
6322789 Trp-Leu-Ser-Val-Ile- Trp-Met-Met-Trp-Tyr-
Trp-Gly-Pro-Ser-Leu 355 HBenv 309-328 Asn-Cys-Thr-Cys-Ile- US
6322789 Pro-Ile-Pro-Ser-Ser- Trp-Ala-Phe-Gly-Lys-
Phe-Leu-Trp-Glu-Trp 356 HBenv 329-358 Ala-Ser-Ala-Arg-Phe- US
6322789 Ser-Trp-Leu-Ser-Leu- Leu-Val-Pro-Phe-Val-
Gln-Trp-Phe-Val-Gly 357 HBc 91-102 Thr-Asn-Met-Gly-Leu- US 6322789
Lys-Phe-Arg-Gln-Leu- Leu-Trp-Leu-Ser-Pro- Thr-Val-Trp-Leu-Ser-
Val-Ile- 358 HBc 128-140 TPPAYRPPNAPIL US 6419931 359 HBenv 360-368
WMMWYWGPSL US 6322789 360 Myobacterium LEDPYEKIGAELVKEV leprae 361
Myobacterium EQIAATAAISAGDQS USSN 11/041893* leprae 362
Myobacterium AGDQSIGDLIAEAMD USSN 11/041893 leprae 363 Myobacterium
VEGAGDTDAIAGRVA USSN 11/041893 leprae 364 Myobacterium
AGGVAVIKAGAATEV USSN 11/041893 leprae 365 Myobacterium
GDEATGANIVKVALE USSN 11/041893 leprae 366 Myobacterium
LQNAASIAGLFLTTE USSN 11/041893 leprae 367 Myobacterium
AGGGVTLLQAAPALD USSN 11/041893 leprae 368 Myobacterium
RVAQIRTEIENSD USSN 11/041893 leprae 369 Myobacterium LLQAAPALDKLKL
USSN 11/041893 leprae 370 Myobacterium PEKTAAPASDPTG USSN 11/041893
leprae 371 Myobacterium LEDPYEKIGAELVKEV USSN 11/041893
tuberculosis 372 Myobacterium EQIAATAAISAGDQS USSN 11/041893
tuberculosis 373 Myobacterium AGDQSIGDLIAEAMD USSN 11/041893
tuberculosis 374 Myobacterium VEGAGDTDAIAGRVA USSN 11/041893
tuberculosis 375 Myobacterium AGGVAVIKAGAATEV USSN 11/041893
tuberculosis 376 Myobacterium GDEATGANTVKVALE USSN 11/041893
tuberculosis 377 Myobacterium LQNAASIAGLFLTTE USSN 11/041893
tuberculosis 378 Myobacterium AGGGVTLLQAAPALD USSN 11/041893
tuberculosis 379 Myobacterium RVQAQIRTEIENSD USSN 11/041893
tuberculosis 380 Myobacterium LLQAAPALDKLKL USSN 11/041893
tuberculosis 381 Myobacterium LPAKFLEGF USSN 11/041893 tuberculosis
382 Myobacterium YLQVPSPSMGRDIKVQFQ USSN 11/041893 tuberculosis 383
Myobacterium GRDIKVQFQSGGNNSPAV USSN 11/041893 tuberculosis 384
Myobacterium GCQTYKEWTLLTSELPQW USSN 11/041893 tuberculosis 385
Myobacterium IPAEFLENF USSN 11/041893 tuberculosis 386 Myobacterium
WPTLIGLAM USSN 11/041893 tuberculosis 387 Myobacterium IPAKFLEGL
USSN 11/041893 tuberculosis 388 Myobacterium MPVGGQSSF USSN
11/041893 tuberculosis 389 Myobacterium MPVGGQSSFY USSN 11/041893
tuberculosis 390 Myobacterium MSQIMYNYPAMMAHAGDM USSN 11/041893
tuberculosis 391 Myobacterium ITYQGWQTQWNQALED USSN 11/041893
tuberculosis 392 Myobacterium ATFAAPVALAA USSN 11/041893
tuberculosis 393 Myobacterium SGATIPQGEQS USSN 11/041893
tuberculosis 394 Myobacterium AVAASNNPELTTLTAALSGQLNPQV USSN
11/041893 tuberculosis/ Mycobacterium bovis 395 Myobacterium
ALSGQLNPQVNLVDTLNSGQY USSN 11/041893 tuberculosis/ Mycobacterium
bovis 396 Myobacterium FSKLPASTIDELKTNSSLLTSILTYH USSN 11/041893
tuberculosis/ Mycobacterium bovis 397 Myobacterium
GNADVVCGGVSTANATVYMIDSVL USSN 11/041893 tuberculosis/ Mycobacterium
bovis 398 Myobacterium AVAASNNPELTTLTAALSGQLNPQV USSN 11/041893
tuberculosis/ Mycobacterium bovis 399 Myobacterium
ALSGQLNPQVNLVDTLNSGQY USSN 11/041893 tuberculosis/ Mycobacterium
bovis 400 Myobacterium FSKLPASTIDELKTNSSLLTSILTYH USSN 11/041893
tuberculosis/ Mycobacterium bovis 401 Myobacterium
GNADVVCGGVSTANATVYMIDSVL USSN 11/041893 tuberculosis/ Mycobacterium
bovis 402 Myobacterium ATTVYMIDSVLMPPA USSN 11/041893 tuberculosis/
Mycobacterium bovis 403 Myobacterium MTEQQWNFAGIEAAASAIQG USSN
11/041893 tuberculosis/ Mycobacterium bovis 404 Myobacterium
EQQWNFAGIEAAA USSN 11/041893 tuberculosis/ Mycobacterium bovis 405
Myobacterium WNFAGIEAA USSN 11/041893 tuberculosis/ Mycobacterium
bovis 406 Myobacterium VQGVQQKWDATATELNNALQ USSN 11/041893
tuberculosis/ Mycobacterium bovis 407 Myobacterium
AWGGSGSEAUQGVQQKWDATATEL USSN 11/041893 tuberculosis/ Mycobacterium
bovis 408 Myobacterium QGVQQKWDATATELNNALQNLART USSN 11/041893
tuberculosis/ Mycobacterium bovis 409 Myobacterium
LARTISEAGQAMASTEGNVTGMFA USSN 11/041893 tuberculosis/ Mycobacterium
bovis 410 Myobacterium EQQWNFAGIEAAA USSN 11/041893 tuberculosis/
Mycobacterium bovis 411 Streptococcus NNNDVNIDRTLVAKQSVVKF USSN
11/041893 mutans 412 Streptococcus QLKTADLPAGRDETTSFVLV USSN
11/041893 mutans 413 Streptococcus LATFNADLTKSVATIYPTVV USSN
11/041893 mutans 414 Chlamydia GDYVFDRI USSN 11/041893 Pneumoniae
415 Chlamydia SLLGNATAL USSN 11/041893 Pneumoniae
416 Chlamydia QAVANGGAI USSN 11/041893 Pneumoniae 417 Chlamydia
RGAFCDKEF USSN 11/041893 Pneumoniae 418 Chlamydia CYGRLYSVKV USSN
11/041893 Pneumoniae 419 Chlamydia KYNEEARKKI USSN 11/041893
Pneumoniae 420 Chlamydia GPKGRHVVI USSN 11/041893 Pneumoniae 421
Escherichia coli TPHPARIGL USSN 11/041893 422 Salmonella
LIQCMLKKTMLSINQ USSN 11/041893 typhimurium 423 Listeria GYKDGNEYI
USSN 11/041893 monocytogenes 424 Borrelia VVKEGTVTLSKNISKSGEVS USSN
11/041893 burgdorferi 425 Lymphocytic FQPQNGQFI USSN 11/041893
choriomeningitis virus 426 Lymphocytic RPQASGVYM USSN 11/041893
choriomeningitis virus 427 Lymphocytic PYIACRTSI USSN 11/041893
choriomeningitis virus 428 Lymphocytic MYPIACRTSI USSN 11/041893
choriomeningitis virus 429 Lymphocytic WPYIACRTSI USSN 11/041893
choriomeningitis virus 430 Lassa Fever virus FGTMPSLTLACLT USSN
11/041893 431 Lassa Fever virus FGMPSLTIACMC USSN 11/041893 432
Lassa Fever virus QGQVDLNDAVQAL USSN 11/041893 433 Lassa Fever
virus QGQADLNDVIQSL USSN 11/041893 434 Lassa Fever virus
ALGMFISDTPGER USSN 11/041893 435 Lassa Fever virus SLGMFVSDTPGER
USSN 11/041893 436 Lassa Fever virus QLDPNAKTWMDIE USSN 11/041893
437 Lassa Fever virus NLIPNAKTWMDIE USSN 11/041893 438 Lassa Fever
virus VWDQFKDLCHMHT USSN 11/041893 439 Lassa Fever virus
VWDQFKDLCHMHT USSN 11/041893 440 Lassa Fever virus IWDEYKHLCRMHT
USSN 11/041893 441 HIV-1 FPVTPQVP USSN 11/041893 442 HIV-1
FPVTPRVPL USSN 11/041893 443 HIV-1 TPQVPLRPM USSN 11/041893 444
HIV-1 YPLTFGWCY USSN 11/041893 445 HIV-1 PLTFGWCYK USSN 11/041893
446 HIV-1 LTFGWCYKL USSN 11/041893 447 HIV-1 EIYKRWIIL USSN
11/041893 448 HIV-1 ILGLNKIV USSN 11/041893 449 HIV-1 ILGLNKIVRMY
USSN 11/041893 450 HBV sAg QAGFFLLTRILTIPQSLD USSN 11/041893 451
HBV sAg SCCCTKPTDGNCTCIPIPSS USSN 11/041893 452 HBV sAg
WEWASVRFSWLS USSN 11/041893 453 HBV sAg LPLLPIFFCLWVYI USSN
11/041893 454 HPV RAHYNIVTF USSN 11/041893 455 HPV
GQAEPDRAHYNIVTFCCKCDSTL USSN 11/041893 RLCVQSTHVDIR 456 EBV
RRIYDLIEL USSN 11/041893 457 EBV RKIYDLIEL USSN 11/041893 458 EBV
FRKAQIQGL USSN 11/041893 459 EBV HRCQAIRK USSN 11/041893 460 EBV
RRARSLSAERY USSN 11/041893 461 EBV DWTGGALLVLYSFALML USSN 11/041893
462 EBV ALLVLYSFAL USSN 11/041893 463 EBV LLVLYSFAL USSN 11/041893
464 EBV VLYSFALML USSN 11/041893 465 EBV LVLGIWIYLLEMLWRRLG USSN
11/041893 466 EBV LIIALYLQQNWWTLLVD USSN 11/041893 467 EBV
IALYLQQNW USSN 11/041893 468 EBV ALYLQQNWW USSN 11/041893 469 EBV
QNWWTLLVD USSN 11/041893 470 EBV LYLQQNWWT USSN 11/041893 471 EBV
LIWMYYHGQRHSDEHHH USSN 11/041893 472 EBV QRHSDEHHEI USSN 11/041893
473 EBV GQRHSDEHH USSN 11/041893 474 EBV YYHGQRHSD USSN 11/041893
475 EBV WMYYHGQRH USSN 11/041893 476 EBV TDDSGHESDSNSNEGRH USSN
11/041893 477 EBV ESDSNSNEG USSN 11/041893 478 EBV DSNSNEGRH USSN
11/041893 479 EBV PHSPSDSAGNDGGPPQL USSN 11/041893 480 EBV
AGNDGGPPQ USSN 11/041893 481 EBV PSDSAGNDG USSN 11/041893 482 EBV
RHSDEHHHDDSLPHPQQ USSN 11/041893 483 EBV EENLLDVFRM USSN 11/041893
484 EBV LVSDYCNVLNKEFTA USSN 11/041893 485 EBV FFIQQAPSNRVMIPAT
USSN 11/041893 486 EBV RVMIPATIGTAMYKL USSN 11/041893 487 EBV
KHSRVRAYTYSKVLG USSN 11/041893 488 EBV RALIKTLPRASYSSH USSN
11/041893 489 EBV ERPIFPHPSKPTFLP USSN 11/041893 490 EBV
EVCQPRKIRPFHPPG USSN 11/041893 491 EBV QKEEAAICGQMDDLSH USSN
11/041893 492 EBV DYCNVLNKEF USSN 11/041893 493 EBV ATIGTAMYK USSN
11/041893 494 EBV FLRGRAYGL USSN 11/041893 495 EBV AVFDRKSDAK USSN
11/041893 496 EBV RRIYLDLIEL USSN 11/041893 497 EBV LLWTLVVLL USSN
11/041893 498 EBV CLGGLLTMV USSN 11/041893 499 EBV IEDPPFNSL USSN
11/041893 500 EBV SSCSSCPLSKI USSN 11/041893 501 EBV TYGPVFMCL USSN
11/041893 502 EBV APENAYQAY USSN 11/041893 503 EBV RAKFKQLL USSN
11/041893 504 EBV GLCTLVAML USSN 11/041893 505 EBV TLDYKPLSV USSN
11/041893 506 EBV QNGALAINTE USSN 11/041893 507 EBV EENLLDFVRF USSN
11/041893 508 EBV HPLTNNLPL USSN 11/041893 509 HCV GYKVLVLNPSVAAT
USSN 11/041893 510 Hantaan virus NAHEGQLVI USSN 11/041893 511
Hantaan virus ISNQEPLKL USSN 11/041893 512 Dengue virus
LIGFRKEIGRMLNIL USSN 11/041893 513 Dengue virus KGPLRMVLAFITFLR
USSN 11/041893 514 Rotavirus RNFDTIRLSFQLVER USSN 11/041893 515
Rotavirus RLSFQLVRPPNMTP USSN 11/041893 516 Rotavirus
VRPPNMTPAVANLF USSN 11/041893 517 Measles virus LSEIKGVIVHRLEAV
USSN 11/041893 518 Trypanosoma IYNVGQVSI USSN 11/041893 cruzi 519
Trypanosoma SHNFTLVASVIIEEA USSN 11/041893 cruzi 520 Trypanosoma
LVASVIIEEAPSGNT USSN 11/041893 cruzi 521 Toxoplasma
TDPGDVVIEELFNRIPETSV USSN 11/041893 gondii 522 Toxoplasma
LQLIRLAASLQHYGLVHA USSN 11/041893 gondii 523 Toxoplasma
IEWIYRRCKNIPQPVRALLEGFLR USSN 11/041893 gondii 524 Babesia bovis
EYLVNKVLYMATMNYKT USSN 11/041893 525 Babesia bovis EAPWYKRWIKKFR
USSN 11/041893 526 Babesia bovis FREAPQATKHFL USSN 11/041893 527
Babesia bovis FREAPQATKHFLDEN USSN 11/041893 528 Babesia bovis
FREAPQATKHFLGEN USSN 11/041893
529 Babesia bovis FVVSLLKKNVVRDPESNDVENFA USSN 11/041893 SQYFYM 530
Babesia bovis VNSEKVDADDAGNAETQQLPDDA USSN 11/041893 ENEVRADD 531
Plasmodium NFVGKFLELQIPGHTDLLHL USSN 11/041893 vivax 532 Plasmodium
FNQLMHVINFHYDLLRANVH USSN 11/041893 vivax 533 Plasmodium
LDMLKKVVLGLWKPLDNIKD USSN 11/041893 vivax 534 Plasmodium
LEYYLREKAKMAGTLIPES USSN 11/041893 vivax 535 Plasmodium
KKIKAFLETSNNKAAAPAQS USSN 11/041893 vivax 536 Plasmodium
SKDQIKKLTSLKNKLERRQN USSN 11/041893 vivax 537 Plasmodium
DPNANPNVDPNANPNV USSN 11/041893 falciparum 538 Plasmodium
FGYRKPLDNIKDNVGKMEDYIKK USSN 11/041893 falciparum 539 Plasmodium
SKLNSLNNPHNVLQNFSVFFNKK USSN 11/041893 falciparum 540 Plasmodium
GYRKPLDNIKDNVGKMEDYIKK USSN 11/041893 falciparum 541 Plasmodium
KLNSLNNPHNVLQNFSVFFNK USSN 11/041893 falciparum 542 Plasmodium
TKILLKHYKGLVKYYNGESSP USSN 11/041893 falciparum 543 Plasmodium
HGTKYLIDGYEEINELLYKLN USSN 11/041893 falciparum 544 Plasmodium
VTHESYQELVKKLEALEDAV USSN 11/041893 falciparum 545 Plasmodium
GLFHKEKMILNEEEITTKGA USSN 11/041893 falciparum 546 Plasmodium
DSNIMNSINNVMDEIDFFEK USSN 11/041893 falciparum 547 Plasmodium
DDYTEYDLTEIDNILVKMFKTN USSN 11/041893 falciparum 548 Plasmodium
LTMSNVKNVSQTNFKSLLRNL USSN 11/041893 falciparum 549 Plasmodium
HTLETVNISDVNDFQISKY USSN 11/041893 falciparum 550 Plasmodium
DDEEDLDEFKPIVQYDNFQD USSN 11/041893 falciparum 551 Plasmodium
EENIGIKELEDLIEKNENL USSN 11/041893 falciparum 552 Plasmodium
DDLDEGIEKSSEELSEEK USSN 11/041893 falciparum 553 Plasmodium
IKKGKKYEKTKDNNF USSN 11/041893 falciparum 554 Plasmodium
DNEILQIVDELSEDITKYFMKL USSN 11/041893 falciparum 555 Plasmodium
EQQQSDLEQERLAKEKLQEQQS USSN 11/041893 falciparum DLEQERRAKEKLQ *The
listed patents and patent applications are hereby expressly
incorporated by reference in their entirety.
[0267] Yet other exemplary TCEs are listed in TABLES 4 as well as
described herein (e.g., SEQ ID NOs: 809-1011 and SEQ ID NO:
1014).
TABLE-US-00005 TABLE 4 HCV T-CELL EPITOPES HLA Re- SEQ Protein
striction Sequence ID NO: Core A2 FLPSDFFPSV 809 Core A2 CLTFGRETV
810 Core A2 VLEYLVSFGV 811 Core A2/A24 EYLVSFGVW 812 Core A2
ILSTLPETTV 813 Core A33/A68 STLPETTVVRR 814 Core A2 AILSKTGDPV 815
Env A2 LLDPRVRGL 816 Env A2 VLQAGFFLL 817 Env A2 FLLTRILTI 818 Env
A2 SLNFLGGTTV 819 Env A2 FLGGTPVCL 820 Env A2 LLLCLIFLL 821 Env A2
LLCLIFLLV 822 Env A2 LLDYQGMLPV 823 Env A2 LVLLDYQGML 824 Env A2
VLLDYQGML 825 Env A2 LLDYQGMLPV 826 Env A2 WLSLLVPFV 827 Env A2
LLVPFVQWFV 828 Env A2 GLSPTVWLSV 829 Env A2 SIVSPFIPLL 830 Env A2
LLPIFFCLWV 831 Env A2 ILSPFFFLPLL 832 x-Protein A2 VLCLRPVGA 833
x-Protein A2 TLPSPSSSA 834 x-Protein A2 EILSLRGLFV 835 x-Protein A2
VLHKRTLGL 836 x-Protein A2 AMSTTDLEA 837 x-Protein A2 CLFKDWEEL 838
Pol A24 LYSSTVPVF 839 Pol A2 GLSRYVARL 840 Pol A2 YMDDVVLGA 841 Pol
A2 FLLSLGIHL 842 Pol A24 KYTSFPWLL 843 Pol A2 ILRGTSFVYV 844 Pol A2
SLYADSPSV 845
[0268] Other exemplary sequences that can be used in part or in
whole as epitopes in the embodiments are also described herein,
e.g., TCE's and BCE's.
[0269] Other exemplary HTL epitopes within a HTL peptide from
tetanus toxoid 830-843 having the sequence:
Gln-Tyr-Ile-Lys-Ala-Asn-Ser-Lys-Phe-Ile-Gly-Ile-Thr-Glu
(QYIKANSKFIGITE) [SEQ ID NO: 556], malaria circumsporozoite 382-398
(KIAKMEKASSVFNVVNS) [SEQ ID NO: 557]; malaria circumsporozoite
378-398 (DIEKKIAKMEKASSVFNVVNS) [SEQ ID NO: 558], malaria
circumsporozoite 326-345 (EYLNKIQNSLSTEWSPCSVT) and ovalbumin
323-336 Ile-Ser-Gln-Ala-Val-His-Ala-Ala-His-Ala-Glu-Ile-Asn-Glu
[SEQ ID NO: 559] and the influenza epitope 307-319
Pro-Lys-Tyr-Val-Lys-Gln-Asn-Thr-Leu-Lys-Leu-Ala-Thr [SEQ ID NO:
560]; Corneybacterium diptheriae dephteria toxin
NLFQVVHWSYNRPAYSPGYV [SEQ ID NO: 561]; Escherichia coli OmpF
FDFGLRPSTAYTKSKAKDVVE [SEQ ID NO: 567]; Escherichia coli OmpF
DEVFATYYFNKNMSTYVDYII [SEQ ID NO: 1379]; Escherichia coli OmpF
NKNMSTYVDYIINQIDSKNK [SEQ ID NO: 568]. In addition, suitable T
helper peptides have been identified as described in pending U.S.
patent application Ser. No. 08/121,101, hereby expressly
incorporated by reference in its entirety.
[0270] Other examples of HTL-inducing peptides are those which are
specific for the antigen (virus or other organism, tumor, etc.)
being targeted by the CTL. For example, several HTL-inducing
peptides specific for HBV have been described, such as
HBc.sub.1-20, having the sequence:
Met-Asp-Ile-Asp-Pro-Tyr-Lys-Glu-Phe-Gly-Ala-Thr-Val-Glu-Leu-Leu-Ser-Phe-L-
eu-Pro [SEQ ID NO: 562]; peptides from the region HBc.sub.50-69,
which has the sequence
Pro-His-His-Tyr-Ala-Leu-Arg-Gln-Ala-Ile-Leu-Cys-Trp-Gly-Glu-Leu-Met-Tyr-L-
eu-Ala [SEQ ID NO: 563], and from the region of HBc.sub.100-139,
including HBc.sub.100-119 having the sequence
Leu-Leu-Trp-Phe-His-Ile-Ser-Cys-Leu-Thr-Phe-Gly-Arg-Glu-Thr-Val-Ile-Glu-T-
yr-Leu [SEQ ID NO: 564] (where Ile.sub.116 is Leu in the HBV adw
subtype), HBc.sub.117-131 having the sequence
Glu-Tyr-Leu-Val-Ser-Phe-Gly-Val-Trp-Ile-Arg-Thr-Pro-Pro-Ala [SEQ ID
NO: 565], and peptide HBc.sub.120-139 having the sequence
Val-Ser-Phe-Gly-Val-Trp-Ile-Arg-Thr-Pro-Pro-Ala-Tyr-Arg-Pro-Pro-Asn-Ala-P-
ro-Ile [SEQ ID NO: 566]. See, Ferrari et al., J. Clin. Invest.
88:214-222 (1991), and U.S. Pat. No. 4,882,145, and U.S. Pat. No.
5,143,726, hereby expressly incorporated by reference in their
entireties.
[0271] The skilled artisan will also appreciate that proteins
containing at least one epitope, such as a TCE, useful in the
embodiments disclose herein can be identified using a variety of
techniques known in the art. Illustrative methods are described in,
for example, Current Protocols in Immunology, Edited by: John E.
Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach,
Warren Strober (2001 John Wiley & Sons, NY, N.Y.) Ausubel et
al. (2001 Current Protocols in Molecular Biology, Greene Publ.
Assoc. Inc. & John Wiley & Sons, Inc., NY, N.Y.); Sambrook
et al. (1989 Molecular Cloning, Second Ed., Cold Spring Harbor
Laboratory, Plainview, N.Y.); Maniatis et al. (1982 Molecular
Cloning, Cold Spring Harbor Laboratory, Plainview, N.Y.).
Illustrative methods useful in this context include intracellular
cytokine staining (ICS), ELISPOT, proliferation assays, cytotoxic T
cell assays including chromium release or equivalent assays, and
gene expression analysis using any number of polymerase chain
reaction (PCR) or RT-PCR based assays.
[0272] Epitopes of the embodiments disclosed herein may be
identified using any number of techniques known in the art, such as
those described by: Lamb J R, et al., (1989) Rev. Infect. Dis.
March-April: Suppl 2:s443-447; Lamb J R, et al. (1987) EMBO J.
6(5):1245-1249; Lamb J R, et al., (1986) Lepr. Rev. Dec.; Suppl
2:131-137; Mehra V, et al., (1986) Proc. Natl. Acad. Sci. USA
83(18): 7013-7; Horsfall A C, et al., (1991) Immunol. Today
12(7):211-3; Rothbard J B et al., (1990) Curr Top Microbiol Immunol
155:143-52; Singh H et al., (2001) Bioinformatics 17:1236-1237;
DeGroot A S, et al. Vaccine 19:4385-4395; DeLalla C, et al., (1999)
J. Immunol. 163:1725-1729; Cochlovius B, et al., (2000) J. Immunol.
165:4731-4741; Consogno G, et al. (2003) Blood 101:1039-1044;
Roberts C G, et al. (1996) AIDS Res. Hum. Retrovir. 12:593-610;
Kwok W, et al. (2001) Trends Immunol. 22:583-588; Novak E J, et
al., (2001) J. Immunol. 166:6665-6670.
[0273] An epitope that is used in some embodiments described herein
may comprise a naturally occurring or naturally processed epitope
as defined using any number of assays known to the skilled artisan
and as described herein. Assays for identifying epitopes are known
to the skilled artisan and are described, for example, in Current
Protocols in Immunology, John E. Coligan, Ada M. Kruisbeek, David
H. Margulies, Ethan M. Shevach, and Warren Strober (Eds), John
Wiley & Sons, New York. N.Y. Epitopes may be identified using
intracellular cytokine staining and flow cytometric analysis such
as described in Hoffmeister B., et al., (2003) Methods.;
29(3):270-281; Maecker H T, et al., (2001) J Immunol Methods
255(1-2):27-40. Similarly, proteins, peptides, overlapping
peptides, or pools of peptides can be used in standard chromium
release and/or proliferation assays to identify epitopes.
[0274] In those cases where antigen-specific T cell lines or clones
are available, for example tumor-infiltrating lymphocytes (TIL) or
virus-specific CTL, these cells can be used to screen for the
presence of the relevant epitopes using target cells prepared with
specific antigens. Such targets can be prepared using random, or
selected synthetic peptide libraries, which would be utilized to
sensitize the target cells for lysis by the CTL. Another approach
to identify the relevant epitope when T cell lines or clones are
available is to use recombinant DNA methodologies. Gene, or
preferably cDNA, libraries from CTL-susceptible targets are first
prepared and transfected into non-susceptible target cells. This
allows the identification and cloning of the gene coding the
protein precursor to the peptide containing the CTL epitope. The
second step in this process is to prepare truncated genes from the
relevant cloned gene, in order to narrow down the region that
encodes for the CTL epitope. This step is optional if the gene is
not too large. The third step is to prepare synthetic peptides of
approximately 10-20 amino acids of length, overlapping by 5-10
residues, which are used to sensitize targets for the CTL. When a
peptide, or peptides, is shown to contain the relevant epitope,
smaller peptides can be prepared to establish the peptide of
minimal size that contains the epitope.
[0275] Alternatively, epitopes may be defined by direct elution of
peptides bound by particular MHC molecule and direct sequencing of
the peptides (see, for example, Engelhard V H, et al., Cancer J.
2000 May; 6 Suppl. 3:S272-80). Briefly, the eluted peptides are
separated using a purification method such as HPLC, and individual
fractions are tested for their capacity to sensitize targets for
CTL lysis or to induce proliferation of cytokine secretion in HTL.
When a fraction has been identified as containing the peptide, it
is further purified and submitted to sequence analysis. The peptide
sequence can also be determined using tandem mass spectrometry. A
synthetic peptide is then prepared and tested with the CTL or HTL
to corroborate that the correct sequence and peptide have been
identified
[0276] Epitopes may also be identified using computer analysis,
such as the Tsites program, which searches for peptide motifs that
have the potential to elicit Th responses. (See, e.g., Rothbard and
Taylor, (1988) EMBO J. 7:93-100; Deavin et al., (1996) Mol.
Immunol. 33:145-155, 1996). CTL peptides with motifs appropriate
for binding to murine and human class I or class II MHC may be
identified according to BIMAS (Parker et al., (19944) J. Immunol.
152:163) and other HLA peptide binding prediction analyses.
Briefly, the protein sequences for example from viral or tumor cell
components are examined for the presence of MHC-binding motifs.
These binding motifs which exist for each MHC allele are conserved
amino acid residues, usually at positions 2 (or 3) and 9 (or 10)
for MHC class I binding peptides of 9-10 residues long. Synthetic
peptides are then prepared of those sequences bearing the MHC
binding motifs, and subsequently are tested for their ability to
bind to MHC molecules. The MHC binding assay can be carried out
either using cells which express high number of empty MHC molecules
(cellular binding assay), or using purified MHC molecules. Lastly,
the MHC binding peptides are then tested for their capacity to
induce a CTL response in naive individuals, either in vitro using
human lymphocytes, or in vivo using HLA-transgenic animals. These
CTL are tested using peptide-sensitized target cells, and targets
that naturally process the antigen, such as viral infected cells or
tumor cells. To further confirm immunogenicity, a peptide may be
tested using an HLA A2 transgenic mouse model and/or any of a
variety of in vitro stimulation assays.
[0277] Epitopes that are used with embodiments described herein may
also be identified using a peptide motif searching program based on
algorithms developed by Rammensee, et al. (Hans-Georg Rammensee,
Jutta Bachmann, Niels Nikolaus Emmerich, Oskar Alexander Bachor,
Stefan Stevanovic: SYFPEITHI: database for MHC ligands and peptide
motifs. Immunogenetics (1999) 50: 213-219); by Parker, et al.
(supra), or using methods such as those described by Doytchinova
and Flower (2002) Immunol Cell Biol. 80(3):270-9 and Blythe M J,
Doytchinova I A, Flower D R. (2002), Bioinformatics 18,
434-439.
[0278] In certain embodiments, an epitope may comprise a variant of
a native epitope. A "variant," as used herein, is a polypeptide (or
a nucleic acid encoding such a polypeptide) that differs from a
native polypeptide in one or more substitutions, deletions,
additions and/or insertions, such that the immunogenicity of the
polypeptide is retained (i.e., the ability of the variant to react
with antigen-specific antisera and/or T-cell lines or clones is not
substantially diminished relative to the native polypeptide). In
other words, the ability of a variant to react with
antigen-specific antisera and/or T-cell lines or clones may be
enhanced or unchanged, relative to the native polypeptide, or may
be diminished by less than 50%, and preferably less than 20%
relative to the native polypeptide. In some embodiments, the
ability of a variant to react with antigen-specific antisera and/or
T-cell lines or clones may be diminished by less than 30%, 25%,
20%, 19%, 18%, 17%, 16%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,
1%, or 0.5%, relative to the native polypeptide. In one embodiment
the ability of a variant to react with antigen-specific antisera
and/or T-cell lines or clones may be enhanced by at least 30%, 25%,
20%, 19%, 18%, 17%, 16%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,
1%, or 0.5%, relative to the native polypeptide. Such variants may
generally be identified by modifying one of the above polypeptide
sequences and evaluating the reactivity of the modified polypeptide
with antisera and/or T-cells as described herein. In some
embodiments, a variant may be identified by evaluating its ability
to bind to a human, murine, or nonhuman primate MHC molecule. In
preferred embodiments, a variant polypeptide has a modification
such that the ability of the variant polypeptide to bind to a class
I or class II MHC molecule is increased relative to that of a wild
type (unmodified) polypeptide. The skilled artisan recognizes that
any number of class I or class II MHC molecules can be used in the
context of the embodiments described herein, for example any HLA
molecule as identified and available from the IMGT/HLA
database.
[0279] In more embodiments, the ability of the variant TCE to bind
to an HLA molecule is increased by at least 2 fold, preferably at
least 3 fold, 4 fold, or 5 fold relative to that of a native
polypeptide. It has been found in some embodiments that a
relatively small number of substitutions (e.g., 1 to 3) within an
epitope can enhance the ability of the epitope to elicit an immune
response. Suitable substitutions may generally be identified by
using computer programs, as described above, and the effect can be
confirmed based on the reactivity of the modified polypeptide with
antisera and/or T-cells as described herein. Accordingly, within
certain preferred embodiments, a variant in which 1 to 3 amino acid
resides within an epitope are substituted such that the ability to
react with antigen-specific antisera and/or T-cell lines or clones
is statistically greater than that for the unmodified polypeptide.
Such substitutions are preferably located within an MHC binding
site of the polypeptide, which may be identified as described
above. Preferred substitutions allow increased binding to MHC class
I or class II molecules.
[0280] The CTL or HTL sequences employed in the compositions and
methods described herein need not be identical to specific amino
acids disclosed in aforementioned disclosures, and can be selected
by a variety of techniques, for example, according to certain
motifs as described above. Therefore, the epitopes may be subject
to various changes, such as insertions, deletions, and
substitutions, either conservative or non-conservative, where such
changes might provide for certain advantages in their use.
Conservative substitutions are discussed above in reference to
TABLE 1. Usually, the portion of the sequence which is intended to
substantially mimic a CTL or HTL stimulating epitope will not
differ by more than about 20% from the corresponding sequence of a
native, or naturally occurring antigen, when known, except where
additional amino acids may be added at either terminus for the
purpose of modifying the physical or chemical properties of the
peptide for, e.g., ease of linking or coupling, and the like. In
those situations where regions of the peptide sequences are found
to be polymorphic among antigen subtypes, it may be desirable to
vary one or more particular amino acids to more effectively mimic
differing CTL or HTL epitopes of different antigen strains.
[0281] In some instances, it may be desirable to combine two or
more epitopes that contribute to stimulating specific CTL or HTL
responses in one or more patients or histocompatibility types. The
epitopes in the composition can be identical or different, and
together they can provide equivalent or greater biological activity
than the parent peptide(s). For example, using the methods
described herein, two or more peptides may define different or
overlapping CTL epitopes from a particular region, e.g., the
peptide region, e.g., HBenv.sub.309-328; peptide region
HBenv.sub.329-349, HBenv.sub.349-368, or peptide region
HBc.sub.91-110, which peptides can be combined in a "cocktail" to
provide enhanced immunogenicity of CTL responses, and peptides can
be combined with peptides having different MHC restriction
elements.
[0282] Compositions
[0283] As will be understood by those skilled in the art, the
nucleic acids of the embodiments disclosed herein can be
single-stranded (coding or antisense), or double-stranded, and may
be a DNA (genomic, cDNA, or synthetic) or RNA molecule. Additional
coding or non-coding sequences may, but need not, be present within
a nucleic acid of the embodiments disclosed herein, and a nucleic
acid may, but need not, be linked to other molecules and/or support
materials.
[0284] Embodiments of the invention also include (a) DNA vectors
that contain any of the foregoing nucleic acid sequence and/or
their complements (i.e., antisense); (b) DNA expression vectors
that contain any of the foregoing nucleic acid sequences
operatively associated with a regulatory element that directs the
expression of the nucleic acid; and (c) genetically engineered host
cells that contain any of the foregoing nucleic acid sequences
operatively associated with a regulatory element that directs the
expression of the coding sequences in the host cell. These
recombinant constructs are capable of replicating autonomously in a
host cell. Alternatively, the recombinant constructs can become
integrated into the chromosomal DNA of a host cell. Such
recombinant polynucleotides typically comprise an HCV genomic or
cDNA polynucleotide of semi-synthetic or synthetic origin by virtue
of human manipulation. Therefore, recombinant nucleic acids
comprising these sequences and complements thereof that are not
naturally occurring are provided.
[0285] Although nucleic acids encoding NS3/4A chimeric peptides or
TCE sequences or nucleic acids having sequences that complement the
NS3/4A chimeric sequences or TCE sequences as they appear in nature
can be employed, they will often be altered, e.g., by deletion,
substitution, or insertion, and can be accompanied by sequence not
present in nature. As used herein, regulatory elements include, but
are not limited to, inducible and non-inducible promoters,
enhancers, operators and other elements known to those skilled in
the art that drive and regulate expression. Such regulatory
elements include, but are not limited to, the cytomegalovirus hCMV
immediate early gene, the early or late promoters of SV40
adenovirus, the lac system, the trp system, the TAC system, the TRC
system, the major operator and promoter regions of phage A, the
control regions offd coat protein, the promoter for
3-phosphoglycerate kinase, the promoters of acid phosphatase, and
the promoters of the yeast-mating factors.
[0286] In addition, recombinant NS3/4A chimeric peptide-encoding
nucleic acid sequences and their complementary sequences can be
engineered so as to modify their processing or expression. For
example, and not by way of limitation, the HCV nucleic acids
described herein can be combined with a promoter sequence and/or
ribosome binding site, or a signal sequence can be inserted
upstream of chimeric polypeptide-encoding sequences so as to permit
secretion of the peptide and thereby facilitate harvesting or
bioavailability. Additionally, a given NS3/4A or TCE nucleic acid
can be mutated in vitro or in vivo, to create and/or destroy
translation, initiation, and/or termination sequences, or to create
variations in coding regions and/or form new restriction sites or
destroy preexisting ones, or to facilitate further in vitro
modification. (See, Examples 1 and 2). Any technique for
mutagenesis known in the art can be used, including but not limited
to, in vitro site-directed mutagenesis. (Hutchinson et al., J.
Biol. Chem., 253:6551 (1978)). The nucleic acids encoding the
chimeric NS3/4A polypeptides described above, can be manipulated
using conventional techniques in molecular biology so as to create
recombinant constructs that express the NS3/4A recombinant
peptides.
[0287] Further, nucleic acids encoding other proteins or domains of
other proteins can be joined to nucleic acids encoding an HCV
peptide so as to create a fusion protein. Nucleotides encoding
fusion proteins can include, but are not limited to, a full length
NS3/4A sequence (SEQ. ID. NO.: 2 or SEQ. ID. NO.: 36), mutant
NS3/4A sequences (e.g., SEQ. ID. NOs.: 3-11 or 40-220) or a peptide
fragment of an NS3/4A sequence fused to an unrelated protein or
peptide, such as for example, polyhistidine, hemagglutinin, an
enzyme, fluorescent protein, or luminescent protein, as discussed
below.
[0288] It was discovered that the construct "NS3/4A-pVAX" was
significantly more immunogenic in vivo than the construct
"NS3-pVAX". Surprisingly, it was also discovered that the
codon-optimized NS3/4A containing construct ("MSLF1-pVAX") was more
immunogenic in vivo than NS3/4A pVAX. The example below describes
these experiments.
Example 3
[0289] To determine whether a humoral immune response was elicited
by the NS3-pVAX and NS3/4A-pVAX vectors, the expression constructs
described in Example 1 were purified using the Qiagen DNA
purification system, according to the manufacturer's instructions
and the purified DNA vectors were used to immunize groups of four
to ten Balb/c mice. The plasmids were injected directly into
regenerating tibialis anterior (TA) muscles as previously described
(Davis et al., Human Gene Therapy 4(6):733 (1993)). In brief, mice
were injected intramuscularly with 50 l/TA of 0.01 mM cardiotoxin
(Latoxan, Rosans, France) in 0.9% sterile NaCl. Five days later,
each TA muscle was injected with 50 .mu.l PBS containing either
rNS3 or DNA.
[0290] Inbred mouse strains C57/BL6 (H-2b), Balb/C (H-2d), and CBA
(H-2k) were obtained from the breeding facility at Mollegard
Denmark, Charles River Uppsala, Sweden, or B&K Sollentuna
Sweden. All mice were female and were used at 4-8 weeks of age. For
monitoring of humoral responses, all mice received a booster
injection of 50 .mu.l/TA of plasmid DNA every fourth week. In
addition, some mice were given recombinant NS3 (rNS3) protein,
which was purified, as described herein. The mice receiving rNS3
were immunized no more than twice. All mice were bled twice a
month.
[0291] Enzyme immunosorbent assays (EIAs) were used to detect the
presence of murine NS3-specific antibodies. These assays were
performed essentially as described (Chen et al., Hepatology 28(1):
219 (1998)). Briefly, rNS3 was passively adsorbed overnight at
4.degree. C. to 96-well microtiter plates (Nunc, Copenhagen,
Denmark) at 1 .mu.g/ml in 50 mM sodium carbonate buffer (pH 9.6).
The plates were then blocked by incubation with dilution buffer
containing PBS, 2% goat serum, and 1% bovine serum albumin for one
hour at 37.degree. C. Serial dilutions of mouse sera starting at
1:60 were then incubated on the plates for one hour. Bound murine
serum antibodies were detected by an alkaline phosphatase
conjugated goat anti-mouse IgG (Sigma Cell Products, Saint Louis,
Mo.) followed by addition of the substrate pNPP (1 tablet/5 ml of
1M Diethanol amine buffer with 0.5 mM MgCl.sub.2). The reaction was
stopped by addition of 1M NaOH and absorbency was read at 405
nm.
[0292] After four weeks, four out of five mice immunized with
NS3/4A-pVAX had developed NS3 antibodies, whereas one out of five
immunized with NS3-pVAX had developed antibodies (FIG. 2). After
six weeks, four out of five mice immunized with NS3/4A-pVAX had
developed high levels (>10.sup.4) of NS3 antibodies (mean levels
10800.+-.4830) and one had a titer of 2160. Although all mice
immunized with NS3-pVAX developed NS3 antibodies, none of them
developed levels as high as that produced by the NS3/4A-pVAX
construct (mean levels 1800.+-.805). The antibody levels elicited
by the NS3/4A fusion construct were significantly higher than those
induced by NS3-pVAX at six weeks (mean ranks 7.6 v.s 3.4,
p<0.05, Mann-Whitney rank sum test, and p<0.01, Students
t-test). Thus, immunization with either NS3-pVAX or NS3/4A-pVAX
resulted in the production of NS3-specific antibodies, but the
NS3/4A containing construct was a more potent immunogen.
[0293] A similar experiment was conducted to compare the
immunogenicity of the NS3/4A-pVAX and MSLF1-pVAX constructs. To
better resemble a future vaccination schedule in humans, however,
the plasmids were delivered to groups of ten mice using a gene gun.
In brief, plasmid DNA was linked to gold particles according to
protocols supplied by the manufacturer (Bio-Rad Laboratories,
Hercules, Calif.). Prior to immunization, the injection area was
shaved and the immunization was performed according to the
manufacturer's protocol. Each injection dose contained 4 .mu.g of
plasmid DNA. Immunizations were performed on weeks 0, 4, and 8.
[0294] The MSLF1 gene was found to be more immunogenic than the
native NS3/4A gene since NS3-specific antibodies were significantly
higher in mice immunized with the MSLF1-pVAX construct at two weeks
after the second and third immunization (TABLE 5). These results
confirmed that the codon-optimized MSLF1-pVAX was a more potent B
cell immunogen than NS3/4A-pVAX.
TABLE-US-00006 TABLE 5 No. of Mean Immunogen Week injections NS3
titre SD Mann-Whitney NS3/4A 2 1 0 0 NS MSLF1 2 1 0 0 NS3/4A 6 2 0
0 p < 0.0002 MSLF1 6 2 2484 3800 NS3/4A 10 3 60 0 p < 0.0001
MSLF1 10 3 4140 4682
[0295] The example below provides more evidence that MSLF-1
(coNS3/4a) produces a strong humoral response.
Example 3A
[0296] To test the intrinsic immunogenicity of the different NS3
genes groups of BALB/c (H-2.sup.d) mice were immunized with the
following vectors: wtNS3/4A (wild type NS3/4a), coNS3/4A
(codon-optimized NS3/4a or MSLF-1), or wtNS3/4A-SFV (wild-type
NS3/4A obtained from SFV expression). Doses of 4 .mu.g DNA was
administered using the gene gun and doses of 10.sup.7 SFV particles
were injected subcutaneously (s.c.). The mice were boosted after
four weeks. The mice immunized with the wtNS3/4A-SFV developed
antibodies already after the first injection suggesting a potent
immunogenicity (FIGS. 3A and 3B). At two weeks after the second
immunization most mice immunized with the coNS3/4A or wtNS3/4A-SFV
vectors had developed mean antibody levels over 10.sup.3 (FIGS. 3A
and 3B). In contrast, none of the mice immunized with the wtNS3/4A
plasmid had developed detectable NS3-specific antibodies at six
weeks (FIGS. 3A and 3B). Thus, both codon optimization and mRNA
amplification by SFV results in an increased B cell immunogenicity
of the NS3/4A gene.
[0297] To indirectly compare the T helper 1 (Th1) and Th2-skewing
of the T cell response primed by wtNS3/4A, coNS3/4A, and
wtNS3/4A-SFV immunizations the levels of NS3-specific IgG1 (Th2)
and IgG2a (Th1) antibodies were analyzed (FIGS. 3A and 3B). The
IgG2a/IgG1-ratio in mice immunized with rNS3 with or without
adjuvant was always <1 regardless of the murine haplotype,
signaling a Th2-dominated response. In contrast, mice immunized
i.m. with the wtNS3 (wild-type NS3), wtNS3/4A, or coNS3/4A
containing plasmids had Th1-skewed Th-cell responses evidenced by
IgG2a/IgG1 ratios of >1 (FIG. 3B). Thus, codon optimization did
not change the IgG subclass distribution. When genetically
immunizing BALB/c mice with NS3/4A using the gene gun the subclass
ratio suggested a mixed Th1/Th2 response (FIG. 3B). It should be
noted that the codon optimized plasmid did not display an increased
in vitro stimulatory capacity of B cells, as compared to the native
plasmid, suggesting that no major immune stimulatory motifs had
been lost or introduced.
[0298] Immunizations using SFV primed a Th1-, or mixed Th1/Th2-like
isotype distribution. The anti-NS3 IgG2a/IgG1-ratio following
wtNS3/4A-SFV immunization ranged from 2.4 to 20 between different
experiments providing evidence of a Th1-like response. This is
similar to the previous experience with SFV vectors where a
Th1-skewed IgG subclass distribution was observed.
[0299] The example below describes experiments that were performed
to determine if mutant NS3/4A peptides, which lack a proteolytic
cleavage site, could elicit an immune response to NS3.
Example 4
[0300] To test if the enhanced immunogenicity of NS3/4A could be
solely attributed to the presence of NS4A, or if the NS3/4A fusion
protein in addition had to be cleaved at the NS3/4A junction,
another set of experiments were performed. In a first experiment,
the immunogenicity of the NS3-pVAX, NS3/4A-pVAX, and mutant NS3/4A
constructs were compared in Balb/c mice. Mice were immunized on
week 0 as described above, and, after two weeks, all mice were bled
and the presence of antibodies to NS3 at a serum dilution of 1:60
was determined (TABLE 6). Mice were bled again on week 4. As shown
in TABLE 6, all the constructs induced an immune response; the
mutant constructs, for example, the NS3/4A-TGT-pVAX vector was
comparable to the NS3-pVAX vector (4/10 vs. 0/10; NS, Fisher's
exact test). The NS3/4A-pVAX vector, however, was more potent than
the mutant constructs.
TABLE-US-00007 TABLE 6 No. of antibody responders to the respective
immunogen after one 100 .mu.g i.m immunization Weeks from 1.sup.st
wild-type mutant example immunization NS3-pVAX NS3/4A-pVAX
NS3/4A-TGT-pVAX 2 0/10 17/20 4/10 4 0/10 20/20 10/10 (<60) (2415
.+-. 3715) (390 .+-. 639) 55% > 10.sup.3 50% > 10.sup.2 10%
> 10.sup.4 10% > 10.sup.3
[0301] During the chronic phase of infection, HCV replicates in
hepatocytes, and spreads within the liver. A major factor in
combating chronic and persistent viral infections is the
cell-mediated immune defense system. CD4+ and CD8+ lymphocytes
infiltrate the liver during the chronic phase of HCV infection, but
they are incapable of clearing the virus or preventing liver
damage. In addition, persistent HCV infection is associated with
the onset of hepatocellular carcinoma (HCC). The examples below
describe experiments that were performed to determine whether the
NS3, NS3/4A, and MSLF1 constructs were capable of eliciting a
T-cell mediated immune response against NS3.
Example 5
[0302] To study whether the constructs described above were capable
of eliciting a cell-mediated response against NS3, an in vivo tumor
growth assay was performed. To this end, an SP2/0 tumor cell line
(SP2/0-Ag14 myeloma cell line (H-2.sup.d)) stably transfected with
the NS3/4A gene was made. The SP2/0 cells were maintained in DMEM
medium supplemented with 10% fetal calf serum (FCS; Sigma
Chemicals, St. Louis, Mo.), 2 mM L-Glutamine, 10 mM HEPES, 100 U/ml
Penicillin and 100 .mu.g/ml Streptomycin, 1 mM non-essential amino
acids, 50 .mu.M .beta.-mercaptoethanol, 1 mM sodium pyruvate
(GIBCO-BRL, Gaithesburgh, Md.). The pcDNA3.1 plasmid containing the
NS3/4A gene was linearized by BglII digestion. A total of 5 .mu.g
linearized plasmid DNA was mixed with 60 .mu.g transfection reagent
(Superfect, Qiagen, Germany) and the mixture was added to a 50%
confluent layer of SP2/0 cells in a 35 mm dish. The transfection
procedure was performed according to manufacturer's protocol.
[0303] Transfected cells were cloned by limiting dilution and
selected by addition of 800 .mu.g geneticin (G418)/ml complete DMEM
medium after 14 days. A stable NS3/4A-expressing SP2/0 clone was
identified using PCR and RTPCR and/or a capture EIA using a
monoclonal antibody to NS3. All EIAs for the detection of murine
NS3 antibodies were essentially performed as follows. In brief,
rNS3 (recombinant NS3 protein produced in E. Coli, dialyzed
overnight against PBS, and sterile filtered) was passively adsorbed
overnight at 4.degree. C. to 96-well microtiter plates (Nunc,
Copenhagen, Denmark) at 1 .mu.g/ml in 50 mM sodium carbonate buffer
(pH 9.6). The plates were then blocked by incubation with dilution
buffer containing PBS, 2% goat serum, and 1% bovine serum albumin
for one hour at +37.degree. C. Serial dilutions of mouse sera
starting at 1:60 were then incubated on the plates for one hour.
Bound murine serum antibodies were detected by an alkaline
phosphatase conjugated goat anti-mouse IgG (Sigma cellproducts,
Saint Louis, Mo. USA) followed by addition of the substrate pNPP (1
tablet/5 ml of 1M Diethanolamine buffer with 0.5 mM MgCl2). The
reaction was stopped by addition of 1M NaOH. Absorbance was then
read at 405 nm.
[0304] The in vivo growth kinetics of the SP2/0 and the
NS3/4A-SP2/0 cell lines were then evaluated in Balb/c mice. Mice
were injected subcutaneously with 2.times.10.sup.6 tumor cells in
the right flank. Each day the size of the tumor was determined
through the skin. The growth kinetics of the two cell lines was
comparable. The mean tumor sizes did not differ between the two
cell lines at any time point, for example. (See TABLE 7).
TABLE-US-00008 TABLE 7 Mouse Tumor Maximum in vivo tumor size at
indicated time point ID cell line 5 6 7 8 11 12 13 14 15 1 SP2/0
1.6 2.5 4.5 6.0 10.0 10.5 11.0 12.0 12.0 2 SP2/0 1.0 1.0 2.0 3.0
7.5 7.5 8.0 11.5 11.5 3 SP2/0 2.0 5.0 7.5 8.0 11.0 11.5 12.0 12.0
13.0 4 SP2/0 4.0 7.0 8.0 10.0 13.0 15.0 16.5 16.5 17.0 5 SP2/0 1.0
1.0 3.0 4.0 5.0 6.0 6.0 6.0 7.0 Group mean 1.92 3.3 5.0 6.2 9.3
10.1 10.7 11.6 12.1 6 NS3/4A-SP2/0 1.0 2.0 3.0 3.5 4.0 5.5 6.0 7.0
8.0 7 NS3/4A-SP2/0 2.0 2.5 3.0 5.0 7.0 9.0 9.5 9.5 11.0 8
NS3/4A-SP2/0 1.0 2.0 3.5 3.5 9.5 11.0 12.0 14.0 14.0 9 NS3/4A-SP2/0
1.0 1.0 2.0 6.0 11.5 13.0 14.5 16.0 18.0 10 NS3/4A-SP2/0 3.5 6.0
7.0 10.5 15.0 15.0 15.0 15.5 20.0 Group mean 1.7 2.7 3.7 5.7 9.4
10.7 11.4 12.4 14.2 p-value of student's 0.7736 0.6918 0.4027
0.7903 0.9670 0.7986 0.7927 0.7508 0.4623 t-test comparison between
group means
[0305] The example below describes experiments that were performed
to determine whether mice immunized with the NS3/4A constructs had
developed a T-cell response against NS3.
Example 6
[0306] To examine whether a T-cell response was elicited by the
NS3/4A immunization, the capacity of an immunized mouse's immune
defense system to attack the NS3-expressing tumor cell line was
assayed. The protocol for testing for in vivo inhibition of tumor
growth of the SP2/0 myeloma cell line in Balb/c mice has been
described in detail previously (Encke et al., J. Immunol. 161:4917
(1998)). Inhibition of tumor growth in this model is dependent on
the priming of cytotoxic T lymphocytes (CTLs). In a first set of
experiments, groups of ten mice were immunized i.m. five times with
one month intervals with either 100 .mu.g NS3-pVAX or 100 .mu.g
NS3/4A-pVAX. Two weeks after the last immunization 2.times.10.sup.6
SP2/0 or NS3/4A-SP2/0 cells were injected into the right flank of
each mouse. Two weeks later the mice were sacrificed and the
maximum tumor sizes were measured. There was no difference between
the mean SP2/0 and NS3/4A-SP2/0 tumor sizes in the NS3-pVAX
immunized mice. (See TABLE 8).
TABLE-US-00009 TABLE 8 Maximum Mouse Dose Tumor tumor size ID
Immunogen (.mu.g) Tumor cell line growth (mm) 1 NS3-pVAX 100 SP2/0
Yes 5 2 NS3-pVAX 100 SP2/0 Yes 15 3 NS3-pVAX 100 SP2/0 No -- 4
NS3-pVAX 100 SP2/0 Yes 6 5 NS3-pVAX 100 SP2/0 Yes 13 Group total
4/5 9.75 .+-. 4.992 6 NS3-pVAX 100 NS3/4A-SP2/0 Yes 9 7 NS3-pVAX
100 NS3/4A-SP2/0 Yes 8 8 NS3-pVAX 100 NS3/4A-SP2/0 Yes 7 9 NS3-pVAX
100 NS3/4A-SP2/0 No -- 10 NS3-pVAX 100 NS3/4A-SP2/0 No -- 3/5 8.00
.+-. 1.00 Note: Statistical analysis (StatView): Student's t-test
on maximum tumor size. P- values <0.05 are considered
significant. Unpaired t-test for Max diam Grouping Variable: Column
1 Hypothesized Difference = 0 Row exclusion: NS3DNA-Tumor-001213
Mean Diff. DF t-Value P-Value NS3-sp2, NS3-spNS3 1.750 5 0.58 0.584
Group Info for Max diam Grouping Variable: Column 1 Row exclusion:
NS3DNA-Tumor-001213 Count Mean Variance Std. Dev. Std. Err NS3-sp2
4 9.750 24.917 4.992 2.496 NS3-spNS3 3 8.000 1.000 1.000 0.57
[0307] To analyze whether administration of different NS3
containing compositions affected the elicitation of a cell-mediated
immune response, mice were immunized with PBS, rNS3, a control DNA,
or the NS3/4A construct, and tumor sizes were determined, as
described above. The NS3/4A construct was able to elicit a T-cell
response sufficient to cause a statistically significant reduction
in tumor size (See TABLE 9).
TABLE-US-00010 TABLE 9 Maximum Mouse Dose Tumor Anti- Tumor tumor
size ID Immunogen (.mu.g) cell line NS3 growth (mm) 1 NS3-pVAX 10
NS3/4A-SP2/0 <60 + 12.0 2 NS3-pVAX 10 NS3/4A-SP2/0 <60 + 20.0
3 NS3-pVAX 10 NS3/4A-SP2/0 60 + 18.0 4 NS3-pVAX 10 NS3/4A-SP2/0
<60 + 13.0 5 NS3-pVAX 10 NS3/4A-SP2/0 <60 + 17.0 Group mean
60 5/5 16.0 .+-. 3.391 6 NS3-pVAX 100 NS3/4A-SP2/0 2160 + 10.0 7
NS3-pVAX 100 NS3/4A-SP2/0 <60 - -- 8 NS3-pVAX 100 NS3/4A-SP2/0
<60 - -- 9 NS3-pVAX 100 NS3/4A-SP2/0 360 - -- 10 NS3-pVAX 100
NS3/4A-SP2/0 <60 + 12.5 Group mean 1260 2/5 11.25 .+-. 1.768 11
NS3/4A-pVAX 10 NS3/4A-SP2/0 <60 + 10.0 12 NS3/4A-pVAX 10
NS3/4A-SP2/0 <60 - -- 13 NS3/4A-pVAX 10 NS3/4A-SP2/0 <60 - --
14 NS3/4A-pVAX 10 NS3/4A-SP2/0 <60 + 13.0 15 NS3/4A-pVAX 10
NS3/4A-SP2/0 <60 + 13.5 Group mean <60 3/5 12.167 .+-. 1.893
16 NS3/4A-pVAX 100 NS3/4A-SP2/0 60 + 10.0 17 NS3/4A-pVAX 100
NS3/4A-SP2/0 360 - -- 18 NS3/4A-pVAX 100 NS3/4A-SP2/0 2160 + 8.0 19
NS3/4A-pVAX 100 NS3/4A-SP2/0 2160 + 12.0 20 NS3/4A-pVAX 100
NS3/4A-SP2/0 2160 + 7.0 Group mean 1380 4/5 9.25 .+-. 2.217 36
p17-pcDNA3 100 NS3/4A-SP2/0 <60 + 20.0 37 p17-pcDNA3 100
NS3/4A-SP2/0 <60 + 7.0 38 p17-pcDNA3 100 NS3/4A-SP2/0 <60 +
11.0 39 p17-pcDNA3 100 NS3/4A-SP2/0 <60 + 15.0 40 p17-pcDNA3 100
NS3/4A-SP2/0 <60 + 18.0 Group mean <60 5/5 14.20 .+-. 5.263
41 rNS3/CFA 20 NS3/4A-SP2/0 >466560 + 13.0 42 rNS3/CFA 20
NS3/4A-SP2/0 >466560 - -- 43 rNS3/CFA 20 NS3/4A-SP2/0 >466560
+ 3.5 44 rNS3/CFA 20 NS3/4A-SP2/0 >466560 + 22.0 45 rNS3/CFA 20
NS3/4A-SP2/0 >466560 + 17.0 Group mean 466560 4/5 17.333 .+-.
4.509 46 PBS -- NS3/4A-SP2/0 <60 + 10.0 47 PBS -- NS3/4A-SP2/0
<60 + 16.5 48 PBS -- NS3/4A-SP2/0 60 + 15.0 49 PBS --
NS3/4A-SP2/0 <60 + 21.0 50 PBS -- NS3/4A-SP2/0 <60 + 15.0 51
PBS -- NS3/4A-SP2/0 <60 - -- Group mean 60 5/6 15.50 .+-. 3.937
Unpaired t-test for Largest Tumor size Grouping Variable: group
Hypothesized Difference = 0 Mean Diff. DF t-Value P-Value
p17-sp3-4, NS3-100-sp3-4 2.950 5 .739 .4933 p17-sp3-4,
NS3/4-10-sp3-4 2.033 6 .628 .5532 p17-sp3-4, NS3-10-sp3-4 -1.800 8
-.643 .5383 p17-sp3-4, NS3/4-100-sp3-4 4.950 7 1.742 .1250
p17-sp3-4, PBS-sp3-4 -1.300 8 -.442 .6700 p17-sp3-4, rNS3-sp3-4
-3.133 6 -.854 .4259 NS3-100-sp3-4, NS3/4-10-sp3-4 -.917 3 -.542
.6254 NS3-100-sp3-4, NS3-10-sp3-4 -4.750 5 -1.811 .1299
NS3-100-sp3-4, NS3/4-100-sp3-4 2.000 4 1.092 .3360 NS3-100-sp3-4,
PBS-sp3-4 -4.250 5 -1.408 .2183 NS3-100-sp3-4, rNS3-sp3-4 -6.083 3
-1.744 .1795 NS3/4-10-sp3-4, NS3-10-sp3-4 -3.833 6 -1.763 .1283
NS3/4-10-sp3-4, NS3/4-100-sp3-4 2.917 5 1.824 .1277 NS3/4-10-sp3-4,
PBS-sp3-4 -3.333 6 -1.344 .2274 NS3/4-10-sp3-4, rNS3-sp3-4 -5.167 4
-1.830 .1412 NS3-10-sp3-4, NS3/4-100-sp3-4 6.750 7 3.416 .0112
NS3-10-sp3-4, PBS-sp3-4 .500 8 .215 .8350 NS3-10-sp3-4, rNS3-sp3-4
-1.333 6 -.480 .6480 NS3/4-100-sp3-4, PBS-sp3-4 -6.250 7 -2.814
.0260 NS3/4-100-sp3-4, rNS3-sp3-4 -8.083 5 -3.179 .0246 PBS-sp3-4,
rNS3-sp3-4 -1.833 6 -.607 .5662 Note: Statistical analysis
(StatView): Student's t-test on maximum tumor size. P-values <
0.05 are considered as significant.
[0308] The example below describes more experiments that were
performed to determine whether the reduction in tumor size can be
attributed to the generation of NS3-specific T-lymphocytes.
Example 7
[0309] In the next set of experiments, the inhibition of SP2/0 or
NS3/4A-SP2/0 tumor growth was again evaluated in NS3/4A-pVAX
immunized Balb/c mice. In mice immunized with the NS3/4A-pVAX
plasmid, the growth of NS3/4A-SP2/0 tumor cells was significantly
inhibited as compared to growth of the non-transfected SP2/0 cells.
(See TABLE 10). Thus, NS3/4A-pVAX immunization elicits CTLs that
inhibit growth of cells expressing NS3/4A in vivo.
TABLE-US-00011 TABLE 10 Maximum Dose Tumor Tumor tumor size Mouse
ID Immunogen (.mu.g) cell line growth (mm) 11 NS3/4A-pVAX 100 SP2/0
No -- 12 NS3/4A-pVAX 100 SP2/0 Yes 24 13 NS3/4A-pVAX 100 SP2/0 Yes
9 14 NS3/4A-pVAX 100 SP2/0 Yes 11 15 NS3/4A-pVAX 100 SP2/0 Yes 25
4/5 17.25 .+-. 8.421 16 NS3/4A-pVAX 100 NS3/4A- No -- SP2/0 17
NS3/4A-pVAX 100 NS3/4A- Yes 9 SP2/0 18 NS3/4A-pVAX 100 NS3/4A- Yes
7 SP2/0 19 NS3/4A-pVAX 100 NS3/4A- Yes 5 SP2/0 20 NS3/4A-pVAX 100
NS3/4A- Yes 4 SP2/0 4/5 6.25 .+-. 2.217 Note: Statistical analysis
(StatView): Student's t-test on maximum tumor size. P- values
<0.05 are considered significant. Unpaired t-test for Max diam
Grouping Variable: Column 1 Hypothesized Difference = 0 Row
exclusion: NS3DNA-Tumor-001213 Mean Diff DF t-Value P-Value
NS3/4-sp2, NS3/4-spNS3 11.000 6 2.526 0.044 Group Info for Max diam
Grouping Variable: Column 1 Row exclusion: NS3DNA-Tumor-001213
Count Mean Variance Std. Dev. Std. Err NS3/4-sp2 4 17.250 70.917
8.421 4.211 NS3/4-spNS3 4 6.250 4.917 2.217 1.109
[0310] In another set of experiments, the inhibition of
NS3/4A-expressing SP2/0 tumor growth was evaluated in MSLF1-pVAX
immunized Balb/c mice. In brief, groups of mice were immunized with
different immunogens (4 .mu.g of plasmid) using a gene gun at weeks
zero, four, eight, twelve, and sixteen. Two weeks after the last
immunization approximately 2.times.10.sup.6 NS3/4A-expressing SP2/0
cells were injected s.c into the right flank of the mouse. The
kinetics of the tumor growth was then monitored by measuring the
tumor size through the skin at days seven, 11, and 13. The mean
tumor sizes were calculated and groups were compared using the
Mann-Whitney non-parametric test. At day 14 all mice were
sacrificed.
[0311] After only a single immunization, tumor inhibiting responses
were observed. (See FIG. 3 and TABLE 11). After two immunizations,
both the NS3/4A-pVAX and MSLF1-pVAX plasmids primed
tumor-inhibiting responses. (See FIG. 4A and TABLE 12). The tumors
were significantly smaller in mice immunized with the MSLF1 gene,
however, as compared to the native NS3/4A gene. After three
injections, both plasmids effectively primed comparable tumor
inhibiting responses. (See FIG. 4B and TABLE 13). These experiments
provided evidence that the MSLF-1 gene was more efficient in
activating tumor inhibiting immune responses in vivo than
NS3/4A-pVAX.
TABLE-US-00012 TABLE 11 Non- Group MSLF1-pVAX1 NS3/4A-pVAX1
immunized MSLF1-pVAX1 -- N.S. p < 0.05 NS3/4A-pVAX1 N.S. -- p
< 0.05 Non-immunized p < 0.05 p < 0.05 --
TABLE-US-00013 TABLE 12 Non- Group MSLF1-pVAX1 NS3/4A-pVAX1
immunized MSLF1-pVAX1 -- p < 0.05 p < 0.01 NS3/4A-pVAX1 p
< 0.05 -- p < 0.01 Non-immunized p < 0.01 p < 0.01
--
TABLE-US-00014 TABLE 13 Non- Group MSLF1-pVAX1 NS3/4A-pVAX1
immunized MSLF1-pVAX1 -- N.S. p < 0.01 NS3/4A-pVAX1 N.S. -- p
< 0.01 Non-immunized p < 0.01 p < 0.01 --
[0312] The example below describes experiments that were performed
to analyze the efficiency of various NS3 containing compositions in
eliciting a cell-mediated response to NS3.
Example 8
[0313] To determine whether NS3-specific T-cells were elicited by
the NS3/4A immunizations, an in vitro T-cell mediated tumor cell
lysis assay was employed. The assay has been described in detail
previously (Sallberg et al., J. Virol. 71:5295 (1997)). In a first
set of experiments, groups of five Balb/c mice were immunized three
times with 100 .mu.g NS3/4A-pVAX i.m. Two weeks after the last
injection the mice were sacrificed and splenocytes were harvested.
Re-stimulation cultures with 3.times.10.sup.6 splenocytes and
3.times.10.sup.6 NS3/4A-SP2/0 cells were set. After five days, a
standard Cr.sup.51-release assay was performed using NS3/4A-SP2/0
or SP2/0 cells as targets. Percent specific lysis was calculated as
the ratio between lysis of NS3/4A-SP2/0 cells and lysis of SP2/0
cells. Mice immunized with NS3/4A-pVAX displayed specific lysis
over 10% in four out of five tested mice, using an effector to
target ratio of 20:1 (See FIGS. 6A and 6B).
[0314] In a next set of experiments, the T cell responses to
MSLF1-pVAX and NS3/4A-pVAX were compared. The ability of the two
plasmids to prime in vitro detectable CTLs were evaluated in
C57/BL6 mice since an H-2b-restricted NS3 epitope had been
previously mapped. Groups of mice were immunized with the two
plasmids and CTLs were detected in vitro using either peptide
coated H-2b expressing RMA-S cells or NS3/4A-expressing EL-4 cells.
Briefly, in vitro stimulation was carried out for five days in
25-ml flasks at a final volume of 12 ml, containing 5 U/ml
recombinant murine IL-2 (mIL-2; R&D Systems, Minneapolis,
Minn.). The restimulation culture contained a total of
40.times.10.sup.6 immune spleen cells and 2.times.10.sup.6
irradiated (10,000 rad) syngenic SP2/0 cells expressing the NS3/4A
protein. After five days in vitro stimulation a standard
.sup.51Cr-release assay was performed. Effector cells were
harvested and a four-hour .sup.51Cr assay was performed in 96-well
U-bottom plates in a total volume of 200 .mu.l. A total of
1.times.10.sup.6 target cells was labeled for one hour with 20
.mu.l of .sup.51Cr (5 mCi/ml) and then washed three times in PBS.
Cytotoxic activity was determined at effector:target (E:T) ratios
of 40:1, 20:1, and 10:1, using 5.times.10.sup.3 51Cr-labeled target
cells/well.
[0315] Alternatively, spleenocytes were harvested from C57BL/6 mice
12 days after peptide immunization and were resuspended in RPMI
1640 medium supplemented with 10% FCS, 2 mM L-Glutamine, 10 mM
HEPES, 100 U/ml Penicillin and 100 .mu.g/ml Streptomycin, 1 mM
non-essential amino acids, 50 .mu.M .beta.-mercaptoethanol, 1 mM
sodium pyruvate. In vitro stimulation was carried out for five days
in 25 ml flasks in a total volume of 12 ml, containing
25.times.10.sup.6 spleen cells and 25.times.10.sup.6 irradiated
(2,000 rad) syngeneic splenocytes. The restimulation was performed
in the presence of 0.05 .mu.M NS3/4A H-2D.sup.b binding peptide
(sequence GAVQNEVTL SEQ. ID. NO.: 37) or a control peptide
H-2D.sup.b peptide (sequence KAVYNFATM SEQ. ID. NO.: 38). After
five days a .sup.51Cr-release assay was performed. RMA-S target
cells were pulsed with 50 .mu.M peptide for 1.5 hrs at +37.degree.
C. prior to .sup.51Cr-labelling, and then washed three times in
PBS. Effector cells were harvested and the four hour .sup.51Cr
assay was performed as described. Cytotoxic activity was determined
at the E:T ratios 60:1, 20:1, and 7:1 with 5.times.10.sup.3
51Cr-labeled target cells/well. By these assays, it was determined
that the MSLF1 gene primed higher levels of in vitro lytic activity
compared to the NS3/4A-pVAX vector. (See FIG. 7A-7L). Similar
results were obtained with both the peptide coated H-2b expressing
RMA-S cells and NS3/4A-expressing EL-4 cells.
[0316] Additional evidence that the codon-optimized MSLF1 gene
primed NS3-specific CTLs more effectively than the native NS3/4A
gene was obtained using flow cytometry. The frequency of
NS3/4A-peptide specific CD8+ T cells were analyzed by ex-vivo
staining of spleen cells from NS3/4A DNA immunized mice with
recombinant soluble dimeric mouse H-2D.sup.b:Ig fusion protein.
Many of the monoclonal antibodies and MHC:Ig fusion proteins
described herein were purchased from BDB Pharmingen (San Diego,
Calif.); Anti-CD16/CD32 (Fc-Block.TM., clone 2.4G2), FITC
conjugated anti-CD8 (clone 53-6.7), FITC conjugated anti-H-2K.sup.b
(clone AF6-88.5), FITC conjugated anti-H-2D.sup.b (clone KH95),
recombinant soluble dimeric mouse H-2D.sup.b:Ig, PE conjugated
Rat-.alpha. Mouse IgG1 (clone X56).
[0317] Approximately, 2.times.10.sup.6 spleen cells resuspended in
100 .mu.l PBS/1% FCS (FACS buffer) were incubated with 1
.mu.g/10.sup.6 cells of Fc-blocking antibodies on ice for 15
minutes. The cells were then incubated on ice for 1.5 hrs with
either 2 .mu.g/10.sup.6 cells of H-2D.sup.b:Ig preloaded for 48
hours at +4.degree. C. with 640 nM excess of NS3/4A derived peptide
(sequence GAVQNEVTL SEQ. ID. NO.: 37) or 2 .mu.g/10.sup.6 cells of
unloaded H-2D.sup.b:Ig fusion protein. The cells were then washed
twice in FACS buffer and resuspended in 100 .mu.l FACS buffer
containing 10 .mu.l/100 .mu.l PE conjugated Rat-.alpha. Mouse IgG1
secondary antibody and incubated on ice for 30 minutes. The cells
were then washed twice in FACS buffer and incubated with 1
.mu.g/10.sup.6 cells of FITC conjugated .alpha.-mouse CD8 antibody
for 30 minutes. The cells were then washed twice in FACS buffer and
resuspended in 0.5 ml FACS buffer containing 0.5 .mu.g/ml of PI.
Approximately 200,000 events from each sample were acquired on a
FACS Calibur (BDB) and dead cells (PI positive cells) were excluded
from the analysis.
[0318] The advantage of quantifying specific CTLs by FACS analysis
is that it bypasses the possible disadvantages of in vitro
expansion of CTLs in vitro prior to analysis. Direct ex-vivo
quantification of NS3-specific CTLs using NS3-peptide loaded
divalent H-2D.sup.b:Ig fusion protein molecules revealed that the
codon optimized MSLF-1 gene primed a effectively primed
NS3-specific CTLs already after two immunizations, whereas the
original NS3/4A gene did not. Thus, the optimized MSLF-1 gene
effectively primes NS3-specific CTLs that are of higher frequency
and of better functionality by all parameters tested, as compared
to the original NS3/4A gene. The example below provides more
evidence that codon optimized NS3/4A efficiently primes NS3
specific cytotoxic T cells.
Example 8A
[0319] Initially, the frequency of NS3-specific CTLs that could be
primed by gene gun immunization using the wtNS3, wtNS3/4A and
coNS3/4A expressing plasmids was determined. The coNS3/4A plasmid
primed higher precursor frequencies of NS3-specific CTL as compared
to the wtNS3 gene enforcing the importance of NS4A (FIG. 8A). No
statistical difference in CTL precursor frequencies was noted
between the wtNS3/4A and coNS3/4A expressing plasmids when analyzed
directly ex vivo (FIG. 8A). A single immunization with the coNS3/4A
plasmid or wtNS3/4A-SFV primed around 1% of peptide-specific CTLs
within two weeks from immunization (FIG. 8A). The specificity of
the detection of NS3-specific CTLs was confirmed by a five-day
restimulation in vitro with the NS3-peptide, by which high
precursor frequencies were observed after immunization with the
coNS3/4A gene (FIG. 8A).
[0320] To directly compare the in vitro lytic activity of the
NS3-specific CTLs primed by different vectors, a standard
.sup.51Cr-release assay was performed after one or two
immunizations. The lytic activity of the in vivo primed CTLs were
assayed on both NS3-peptide loaded H-2D.sup.b expressing RMA-S
cells and EL-4 cells stably expressing NS3/4A. After one dose, the
coNS3/4A plasmid and the wtNS3/4A-SFV vector was clearly more
efficient than the wtNS3/4A plasmid in priming CTLs that lysed
NS3-peptide coated target cells (FIGS. 9A and 9B). Thus, the CTL
priming event was enhanced by codon optimization or mRNA
amplification of the NS3/4A gene. The difference was less clear
when using the NS3/4A-expressing EL-4 cells presumably since this
assay is less sensitive (FIGS. 9A and 9B). After two immunizations
all NS3/4A vectors seemed to prime NS3-specific CTLs with a similar
efficiency (FIG. 9B). However, two immunizations with any of the
NS3/4A-containing vectors were clearly more efficient in priming
NS3-specific CTLs as compared to the plasmid containing only the
wtNS3 gene (FIG. 9B), which is fully consistent with the CTL
precursor analysis and previous observations. Thus, codon
optimization or mRNA amplification of the NS3/4A gene more rapidly
primes NS3-specific CTLs.
[0321] Analysis of the inhibition of tumor growth in vivo in BALB/c
mice using SP2/0 myeloma cells, or in C57BL/6 mice using EL-4
lymphoma cells, expressing an HCV viral antigen is recognized by
those in the field to represent the in vivo functional HCV-specific
immune response. (See Encke J et al., J Immunol 161: 4917-4923
(1998)). An SP2/0 cell line stably expressing NS3/4A has previously
been described (see Frelin L et al., Gene Ther 10: 686-699 (2003))
and an NS3/4A expressing EL-4 cell line was characterized as
described below.
[0322] To confirm that inhibition of tumor growth using the
NS3/4A-expressing EL-4 cell line is fully dependent on an
NS3/4A-specific immune response a control experiment was performed.
Groups of ten C57BL/6 mice were either left nonimmunized, or
immunized twice with the coNS3/4A plasmid. Two weeks after the last
immunization the mice were challenged with an s.c. injection of
10.sup.6 native EL-4 or NS3/4A-expressing EL-4 cells (NS3/4A-EL-4).
An NS3/4A-specific immune response was required for protection,
since only the immunized mice were protected against growth of the
NS3/4A-EL-4 cell line (FIG. 10A). Thus, this H-2.sup.b-restricted
model behaves similarly to the SP2/0 H-2.sup.d restricted
model.
[0323] Immunizations with recombinant NS3 protein provided evidence
that both NS3/4A-specific B cells and CD4+ T cells were not of a
pivotal importance in protection against tumor growth. In vitro
depletion of CD4+ or CD8+ T cells of splenocytes from coNS3/4A
plasmid immunized H-2.sup.b mice provided evidence that CD8+ T
cells were the major effector cells in the .sup.51Cr-release assay.
To define the in vivo functional anti-tumor effector cell
population, CD4+ or CD8+ T cells in mice immunized with the
coNS3/4A plasmid one week prior to, and during, challenge with the
NS3/4A-EL-4 tumor cell line were selectively depleted. Analysis by
flow cytometry revealed that 85% of CD4+ and CD8+ T cells had been
depleted, respectively. This experiment revealed that in vivo
depletion of CD4+ T cells had no significant effect on the tumor
immunity (FIG. 10B). In contrast, depletion of CD8+ T cells in vivo
significantly reduced the tumor immunity (p<0.05, ANOVA; FIG.
10B). Thus, as expected, NS3/4A-specific CD8+ CTLs seems to be the
major protective cell at the effector stage in the in vivo model
for inhibition of tumor growth.
[0324] The tumor challenge model was then used to evaluate how
effective the different immunogens were in priming a protective
immunity against growth of NS3/4A-EL-4 tumor cells in vivo. To
ensure that the effectiveness of the priming event was studied, all
mice were immunized only once. Fully consistent with the in vitro
CTL data did we find that only vectors containing NS3/4A were able
to rapidly prime protective immune responses as compared to the
immunized with the empty pVAX plasmid (p<0.05, ANOVA; FIG. 11).
However, this was dependent on NS4A but independent of either codon
optimization or mRNA amplification, suggesting that C57BL/6 mice
are quite easily protected against tumor growth using genetic
immunization.
[0325] To further clarify the prerequisites for priming of the in
vivo protective CD8+ CTL responses additional experiments were
performed. First, C57BL/6 mice immunized with the NS3-derived CTL
peptide were not protected against growth of NS3/4A-EL-4 tumors
(FIG. 11). Second, immunization with recombinant NS3 in adjuvant
did not protect against tumor growth (FIG. 11). NS3-derived CTL
peptide effectively primes CTLs in C57BL/6 mice and rNS3 in
adjuvant primes high levels of NS3-specific T helper cells. Thus,
an endogenous production of NS3/4A seems to be needed to prime in
vivo protective CTLs. To further characterize the priming event,
groups of B cell (.mu.MT) or CD4 deficient C57BL/6 mice were
immunized once with the coNS3/4A gene using gene gun, and were
challenged two weeks later (FIG. 11). Since both lineages were
protected against tumor growth we conclude that neither B cells nor
CD4+ T cells were required for the priming of in vivo functional
NS3/4A-specific CTLs (FIG. 11). In conclusion, the priming of in
vivo tumor protective NS3/4A-specific CTLs in C57BL/6 mice requires
NS4A and an endogenous expression of the immunogen. In C57BL/6 mice
the priming is less dependent on the gene delivery route or
accessory cells, such as B cells or CD4+ T cells. The fact that the
priming of in vivo functional CTL by the coNS3/4A DNA plasmid was
independent of CD4+ T helper cells may help to explain the speed by
which the priming occurred.
[0326] Repeated experiments in C57BL/6 mice using the NS3/4A-EL-4
cell line have shown that protection against tumor growth is
obtained already after the first immunization with the NS3/4A gene,
independent of codon optimization or mRNA amplification. Also,
after two injections the immunity against NS3/4A-EL-4 tumor growth
was even further enhanced, but only when NS4A was present. Thus,
this model may therefore not be sufficiently sensitive to reveal
subtle differences in the intrinsic immunogenicity of different
immunogens.
[0327] To better compare the immunogenicity of the wtNS3/4A and the
coNS3/4A DNA plasmids, additional experiments were performed in
H-2.sup.d mice, were at least two immunizations seemed to be
required for a tumor protective immunity. It is important to
remember that the IgG subclass distribution obtained after gene gun
immunization with the NS3/4A gene in BALB/c mice suggested a mixed
Th1/Th2-like response. Thus, it was possible that a Th2-like
immunization route (gene gun) in the Th2-prone BALB/c mouse strain
may impair the ability to prime in vivo effective CTL
responses.
[0328] Groups of ten BALB/c mice were immunized once, twice, or
thrice with 4 g of the respective DNA plasmid using the gene gun
(FIGS. 12A-12C). The mice were challenged two weeks after the last
injection. Accordingly, these experiments provided more evidencer
that the coNS3/4A plasmid primed an in vivo functional
NS3/4A-specific tumor inhibiting immunity more rapidly than the
wild type plasmid (FIGS. 12A-12C). Two doses of the coNS3/4A primed
a significantly better NS3/4A-specific tumor inhibiting immunity as
compared to the wtNS3/4A plasmid (p<0.05, ANOVA; FIGS. 12A-12C).
After three doses the tumor inhibiting immunity was the same. Thus,
the data above verified that the codon optimization of the NS3/4A
gene primes NS3-specific CTLs more rapidly.
[0329] As set forth herein, the NS3/4A gene can be used as a
vaccine. Although it had been determined that NS3/4A quickly primed
in vivo functional CTLs, the effect of therapeutic immunization
after the injection of tumor cells was analyzed next. Groups of ten
C57BL/6 mice were challenged with 10.sup.6 NS3/4A-EL-4 tumor cells.
One group was immunized transdermally with of 4 .mu.g coNS3/4A at
six days, and another group at 12 days, after tumor challenge.
After the therapeutic vaccination both groups had significantly
smaller tumors as compared to the nonimmunized control group
(p<0.01, respectively, ANOVA; FIG. 13). This confirms that the
vaccine rapidly primes CTLs, which are able to home to and
infiltrate the NS3/4A-expressing tumors. Thus, gene gun
immunization with the coNS3/4A plasmid also works as a therapeutic
vaccine. That is, gene gun immunization using the coNS3/4A gene six
to 12 days after inoculation of NS3/4A-expressing tumor cells
significantly inhibited tumor growth. Overall, a rapid priming of
HCV NS3-specific immune responses that are functional in vivo are
generated by either DNA based immunization with a codon optimized
gene or by mRNA amplification by the SFV replicon. By using these
approaches, one can prepare very effective vaccines for the
treatment and prevention of chronic HCV infections. The next
example described in greater detail some of the materials and
methods used in the experiments described herein.
Example 8B
Mice
[0330] Inbred BALB/c (H-2.sup.d) and C57BL/6 (H-2.sup.b) mice were
obtained from commercial vendors (Mollegard, Denmark). B cell
(.mu.MT) deficient mice were kindly provided by Dr Karin Sandstedt,
Karolinska Institutet, Sweden. CD4 deficient C57BL/6 mice were
obtained from the breeding facility at the Microbiology and
Tumorbiology Centre, Karolinska Institutet. All mice were female
and were used at 4-8 weeks of age at the start of the
experiments.
Recombinant NS3 ATPase/Helicase Domain Protein
[0331] The recombinant NS3 (rNS3) protein was kindly provided by
Darrell L. Peterson, Department of Biochemistry, Commonwealth
University, Va. The production of recombinant NS3 protein (not
including NS4A) in E. Coli has been described in the field. Prior
to use the rNS3 protein was dialyzed over night against PBS and
sterile filtered.
Generation of a Synthetic Codon Optimized (Co) NS3/4A Gene
[0332] The sequence of the previously isolated and sequenced unique
wtNS3/4A gene was analyzed for codon usage with respect to the most
commonly used codons in human cells. A total of 435 nucleotides
were replaced to optimize codon usage for human cells. The sequence
was sent to Retrogen Inc (San Diego, Calif.) for generation of a
full-length synthetic coNS3/4A gene. The coNS3/4A gene had a
sequence homology of 79% with the region at nucleotide positions
3417-5475 of the HCV-1 reference strain. A total of 433 nucleotides
differed. On an amino acid level the homology with the HCV-1 strain
was 98% (15 amino acids differed).
[0333] The full-length codon optimized 2.1 kb DNA fragment of the
HCV genotype 1b corresponding to the amino acids 1007 to 1711
encompassing the NS3 and NS4A. NS3/NS4A gene fragment was inserted
into a Bam HI and Xba I digested pVAX vector (Invitrogen, San
Diego) to give the coNS3/4A-pVAX plasmid. The expression construct
was sequenced to ensure correct sequence and reading frame. The
protein expression was analysed by an in vitro transcription and
translation assay. Plasmids were grown in competent TOP 10 E. Coli.
(Invitrogen). Plasmid DNA used for in vivo injection, was purified
by using Qiagen DNA purification columns according to the
manufacturers instructions (Qiagen GmbH, Hilden, FRG). The
concentration of the resulting plasmid DNA was determined
spectrophotometrically (Dynaquant, Pharmacia Biotech, Uppsala,
Sweden). Purified DNA was dissolved in sterile phosphate buffer
saline (PBS) at concentrations of 1 mg/ml.
In Vitro Translation Assay
[0334] To ensure that the wtNS3/4A and coNS3/4A genes were intact
and could be translated, an in vitro transcription assay is using
the prokaryotic T7 coupled reticulocyte lysate system (TNT;
Promega, Madison, Wis.) was performed. To compare the translation
efficiency from the two plasmids the amount input DNA was diluted
in serial dilutions (6 ng to 1 ng) prior to addition to the TNT
assay.
Transient Transfections
[0335] HepG2 cells were transiently transfected by standard
protocols. In brief, HepG2 cells were plated into 2.5 cm.sup.2
wells (0.5.times.10.sup.6) in DMEM medium the day before
transfection. Two .mu.g of each plasmid DNA construct (wtNS3/4A and
coNS3/4A) was transfected into HepG2 cells using Fugene 6
Transfection Reagent (Roche). After transfection, the HepG2 cells
were incubated for 24-48 hrs.
Protein Sample Preparation and Analysis
[0336] Cell lysates were analysed by immunoprecipitation followed
by SDS-PAGE. In brief, transient transfected HepG2 cells were lysed
in RIPA buffer (0.15M NaCl, 50 mM Tris, 1% Triton-X 100, 1%
Na-deoxycholate and 1% SDS). The cell lysates were
immunoprecipitated with protein A sepharose and anti-NS3 polyclonal
antibody overnight at 4.degree. C. The washed pellets were
re-suspended in SDS sample buffer, heated at 100.degree. C. for 5
minutes prior to SDS-PAGE analysis on 4-12% Bis-Tris gel
(Invitrogen) and electrotransferred onto Nitrocellulose
membranes.
Analysis of NS3 Protein Expression
[0337] Detection of NS3 protein was done according to
manufacturer's protocol by using a chemiluminiscence-linked Western
blot kit (WesternBreeze; Invitrogen). NS3 protein expression was
detected and quantified as a chemiluminiscent signal by using an
NS3-specific polyclonal antibody. Chemiluminiscent signals were
detected by using the GeneGnome (Syngene, Cambridge, UK).
Quantification of chemiluminiscence Western blots was performed on
GeneGnome and units of intensity from each protein band was
calculated and compared to a standard curve of rNS3.
Semliki Forest Virus (SFV) Vectors
[0338] Baby Hamster Kidney (BHK)-21 cells were maintained in
complete BHK medium supplemented with 5% FCS, 10% tryptose
phosphate broth, 2 mM glutamine, 20 mM Hepes and antibiotics
(streptomycin 10 .mu.g/ml and penicillin 100 IU/ml).
[0339] The wtNS3/4A gene was isolated by PCR as Spe1-BStB1 fragment
and inserted into the Spe1-BstB1 site of pSFV10Enh containing a 34
amino acid long translational enhancer sequence of capsid followed
by the FMDV 2a cleavage peptide. Packaging of recombinant RNA into
rSFV particles was done using a two-helper RNA system. Indirect
immunofluorescence of infected BHK cells was performed to determine
the titre of the recombinant virus stocks.
Immuno Fluorescence
[0340] BHK cells were transient transfected with coNS3/4A-pVAX1
according to standard techniques using Lipofectamine plus reagent
(Invitrogen) or infected by rSFV. NS3 protein was detected by
indirect immunofluorescence.
Immunization Protocols
[0341] Groups (5-10 mice/group) of female BALB/c (H-2.sup.d) or
C57BL/6 (H-2.sup.b) mice, 4-8 weeks old, were immunized by needle
injections of 100 .mu.g of plasmid DNA encoding individual or
multiple HCV proteins. Plasmid DNA in PBS was given intramuscularly
(i.m.) in the tibialis anterior (TA) muscle. Where indicated in the
text, the mice were injected i.m. with 50 .mu.L/TA of 0.01 mM
cardiotoxin (Latoxan, Rosans, France) in 0.9% sterile saline NaCl,
five days prior to DNA immunization. The mice were boosted at
four-week intervals.
[0342] For gene gun based immunizations, plasmid DNA was linked to
gold particles (1 .mu.m) according to protocols supplied by the
manufacturer (Bio-Rad Laboratories, Hercules, Calif.). Prior to
immunization the abdominal injection area was shaved and the
immunization was performed according to the manufacturer's protocol
at a helium discharge pressure of 500 psi. Each injection dose
contained 4 .mu.g of plasmid DNA. The mice were boosted with the
same dose at monthly intervals.
[0343] For rSFV particle immunizations, mice were immunized
subcutaneously, in the base of the tail, with 1.times.10.sup.7
virus particles diluted in PBS (wtNS3/4A-SFV), in a final volume of
100 .mu.l. Peptide immunization was performed by subcutaneous
immunization in the base of the tail with 100 .mu.g peptide mixed
1:1 in complete Freunds adjuvant.
ELISA for Detection of Murine Anti-HCV NS3 Antibodies
[0344] Serum for antibody detection and isotyping was collected
every second or fourth week after the first immunization by
retroorbital bleeding of isofluorane-anesthetized mice. The enzyme
immuno assays were performed as previously described.
Cell Lines
[0345] The SP2/0-Ag14 myeloma cell line (H-2.sup.d) was maintained
in DMEM medium supplemented with 10% fetal calf serum (FCS; Sigma
Chemicals, St. Louis, Mo.), 2 mM L-Glutamin, 10 mM HEPES, 100 U/ml
Penicillin and 100 .mu.g/ml Streptomycin, 1 mM non-essential amino
acids, 50 .mu.M .beta.-mercaptoethanol, 1 mM sodium pyruvate
(GIBCO-BRL, Gaithesburgh, Md.). SP2/0-Ag14 cells with stable
expression of NS3/4A were maintained in 800 .mu.g geneticin
(G418)/ml complete DMEM medium.
[0346] The EL-4 lymphoma (H-2.sup.b) cells were maintained in RPMI
1640 medium supplemented with 10% FCS, 10 mM HEPES, 1 mM sodium
pyruvate, 1 mM non-essential amino acids, 50 .mu.M
.beta.-mercaptoethanol, 100 U/ml Penicillin and 100 .mu.g/ml
Streptomycin (GIBCO-BRL). EL-4 cells with stable expression of
NS3/4A were generated by transfection of EL-4 cells with the
linearized NS3/4A-pcDNA3.1 plasmid using the SuperFect (Qiagen
GmbH, Hilden, FRG) transfection reagent. The transfection procedure
was performed according to manufacturer's protocol. Transfected
cells were cloned by limiting dilution and selected by addition of
800 .mu.g geneticin (G418)/ml complete RPMI 1640 medium.
[0347] RMA-S cells (a kind gift from Professor Klas Karre,
Karolinska Institutet, Sweden) were maintained in RPMI 1640 medium
supplemented with 5% FCS, 2 mM L-Glutamin, 100 U/ml Penicillin and
100 .mu.g/ml Streptomycin. All cells were grown in a humidified
37.degree. C., 5% CO.sub.2 incubator.
In Vivo Depletion of T Cells
[0348] CD4 and CD8 T cell subpopulations were depleted in vivo by
intraperitoneal injection of purified hybridoma supernatant. A
total of 0.4 mg per mouse per injection of anti-CD4 (clone GK1.5)
or anti-CD8 (clone 53-6.7) was injected on days -3, -2, and -1
before tumor challenge, and on days 3, 6, 10, and 13 after
challenge. Flow cytometric analysis of peripheral blood mononuclear
cell populations at days 0, 3, 6, 10, and 13 demonstrated that more
than 85% of the CD4 and CD8 T cells were depleted.
In Vivo Challenge with the NS3/4A-Expressing Tumor Cells
[0349] In vivo challenge of immunized mice with the
NS3/4A-expressing SP2/0 myeloma or EL-4 lymphoma cell line was
performed according to the method described by Encke et al., supra.
In brief, groups of BALB/c or C57BL/6 mice were immunized with
different immunogens at weeks zero, four, and eight as described.
Two weeks after the last immunisation 1.times.10.sup.6
NS3/4A-expressing SP2/0 or EL-4 cells were injected subcutaneously
in the right flank. The kinetics of the tumor growth was determined
by measuring the tumor size through the skin at days six to 20.
Kinetic tumor development in two groups of mice was compared using
the area under the curve (AUC). The mean tumor sizes were compared
using the analysis of variance (ANOVA) test. At day 20 all mice
were sacrificed.
[0350] To test the therapeutic effect of the vaccines groups of
mice were inoculated with the tumor cells as described above. After
six or 12 days the mice were immunized once. The tumor growth was
monitored from day 6 to day 20.
Antibodies and MHC:Ig Fusion Protein
[0351] All monoclonal antibodies and MHC:Ig fusion proteins were
purchased from BDB Pharmingen (San Diego, Calif.); Anti-CD16/CD32
(Fc-Block.TM., clone 2.4G2), FITC conjugated anti-CD8 (clone
53-6.7), Cy-Chrome conjugated anti-CD4 (clone RM4-5), FITC
conjugated anti-H-2D.sup.b (clone KH95), recombinant soluble
dimeric mouse H-2D.sup.b:Ig, PE conjugated Rat-.alpha. Mouse IgG1
(clone X56).
Detection of NS3/4A-Specific CTL Activity
[0352] Spleen cells from DNA or rSFV immunized C57BL/6 mice were
resuspended in complete RPMI 1640 medium supplemented with 10% FCS,
2 mM L-Glutamine, 10 mM HEPES, 100 U/ml Penicillin and 100 .mu.g/ml
Streptomycin, 1 mM non-essential amino acids, 50 .mu.M
.beta.-mercaptoethanol, 1 mM sodium pyruvate. In vitro stimulation
was carried out for five days in 25-ml flasks at a final volume of
12 ml, containing 5 U/ml recombinant murine IL-2 (mIL-2; R&D
Systems, Minneapolis, Minn., USA). The restimulation culture
contained a total of 25.times.10.sup.6 immune spleen cells and
2.5.times.10.sup.6 irradiated (10,000 rad) syngenic EL-4 cells
expressing the NS3/4A protein. After five days in vitro stimulation
a standard .sup.51Cr-release assay was performed. Effector cells
were harvested and a four-hour .sup.51Cr assay was performed in
96-well U-bottom plates in a total volume of 200 .mu.l. A total of
1.times.10.sup.6 target cells (NS3/4A expressing EL-4 cells) was
labelled for one hour at +37.degree. C. with 20 .mu.l of .sup.51Cr
(5 mCi/ml) and then washed three times in PBS. Different numbers of
effectors and .sup.51Cr-labeled target cells (5.times.10.sup.3
cells/well) were added to wells at effector:target (E:T) ratios of
60:1, 20:1, and 7:1. The level of cytolytic activity was determined
after incubation of effectors and targets for 4 hour at +37.degree.
C. 100 .mu.l supernatant was harvested and the radioactivity was
measured with a .gamma.-counter.
[0353] Splenocytes from DNA or rSFV immunised mice were harvested
from C57BL/6 mice and were resuspended in complete RPMI 1640 medium
as previously described. In brief, in vitro stimulation was carried
out for five days by mixing 25.times.10.sup.6 spleen cells and
25.times.10.sup.6 irradiated (2,000 rad) syngeneic splenocytes. The
restimulation was performed in the presence of 0.05 .mu.M NS3/4A
H-2D.sup.b binding peptide (sequence GAVQNEVTL (Seq. Id. No. 37)).
After restimulation, a four hour .sup.51Cr-release assay was
performed using .sup.51Cr-labelled peptide pulsed RMA-S cells as
targets. Cytotoxic activity was determined at the E:T ratios 60:1,
20:1, and 7:1.
[0354] Results were expressed according to the formula: percent
specific lysis=(experimental release-spontaneous release)/(maximum
release-spontaneous release). Experimental release is the mean
counts/minute released by the target cells in presence of effector
cells. Maximum release is the radioactivity released after lysis of
target cells with 10% Triton X-100. Spontaneous release is the
leakage of radioactivity into the medium of target cells.
[0355] In vitro T-cell depletion experiments were conducted by
incubating effector cells with either an anti-CD4, or anti-CD8,
monoclonal antibody containing hybridoma supernatant (clone RL
172.4; anti-CD4, or clone 31M; anti-CD8) for 30 minutes at
4.degree. C. The cells were then washed and incubated at 37.degree.
C. for 1 hr with complement (1/20 dilution of low toxicity rabbit
complement; Saxon, UK) before performing the CTL assay described
above.
Quantification of NS3/4A-Specific CTLs by Flow Cytometry
[0356] The frequency of NS3-peptide specific CD8+ T cells were
analysed by ex-vivo staining of spleen cells from DNA or rSFV
immunized mice with recombinant soluble dimeric mouse H-2D.sup.b:Ig
fusion protein as previously described. In brief, spleen cells were
resuspended in PBS/1% FCS (FACS buffer) and incubated with
Fc-blocking antibodies. Cells were then washed and incubated with
H-2D.sup.b:Ig preloaded with NS3/4A derived peptide. Afterwards,
cells were washed and incubated with PE conjugated Rat-.alpha.
Mouse IgG1 antibody, FITC conjugated .alpha.-mouse CD8 antibody and
Cy-Chrome .alpha.-mouse CD4 antibody. After washing, the cells were
diluted in FACS buffer containing Propidium Iodide (PI).
Approximately 200,000 total events from each sample were acquired
on a FACSCalibur (BDB) and dead cells (PI positive cells) were
excluded in the analysis.
Statistical Analysis
[0357] Fisher's exact test was used for frequency analysis and
Mann-Whitney U-test was used for comparing values from two groups.
Kinetic tumor development in two groups of mice was compared using
the area under the curve (AUC). AUC values were compared using
analysis of variance (ANOVA). The calculations were performed using
the Macintosh version of the StatView software (version 5.0).
[0358] The compositions described herein may contain other
ingredients or compounds in addition to nucleic acids and/or
polypeptides, including, but not limited to, various other
peptides, adjuvants, binding agents, excipients such as stabilizers
(to promote long term storage), emulsifiers, thickening agents,
salts, preservatives, solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents and the like. See e.g., U.S. application Ser. No.
09/929,955 and U.S. application Ser. No. 09/930,591. These
compositions are suitable for treatment of animals, particularly
mammals, either as a preventive measure to avoid a disease or
condition or as a therapeutic to treat animals already afflicted
with a disease or condition.
[0359] Many other ingredients may also be present in the
compositions provided herein. For example, the adjuvant and antigen
can be employed in admixture with conventional excipients (e.g.,
pharmaceutically acceptable organic or inorganic carrier substances
suitable for parenteral, enteral (e.g., oral) or topical
application that do not deleteriously react with the adjuvant
(e.g., ribavirin) and/or antigen). Suitable pharmaceutically
acceptable carriers include, but are not limited to, water, salt
solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols,
polyetylene glycols, gelatine, carbohydrates such as lactose,
amylose or starch, magnesium stearate, talc, silicic acid, viscous
paraffin, perfume oil, fatty acid monoglycerides and diglycerides,
pentaerythritol fatty acid esters, hydroxy methylcellulose,
polyvinyl pyrrolidone, etc. Many more suitable carriers are
described in Remmington's Pharmaceutical Sciences, 15th Edition,
Easton:Mack Publishing Company, pages 1405-1412 and 1461-1487(1975)
and The National Formulary XIV, 14th Edition, Washington, American
Pharmaceutical Association (1975).
[0360] The gene constructs described herein, in particular, can be
formulated with or administered in conjunction with agents that
increase uptake and/or expression of the gene construct by the
cells relative to uptake and/or expression of the gene construct by
the cells that occurs when the identical genetic vaccine is
administered in the absence of such agents. Such agents and the
protocols for administering them in conjunction with gene
constructs are described in PCT Patent Application Serial Number
PCT/US94/00899 filed Jan. 26, 1994. Examples of such agents
include: CaPO.sub.4, DEAE dextran, anionic lipids; extracellular
matrix-active enzymes; saponins; lectins; estrogenic compounds and
steroidal hormones; hydroxylated lower alkyls; dimethyl sulfoxide
(DMSO); urea; and benzoic acid esters anilides, amidines, urethanes
and the hydrochloride salts thereof, such as those of the family of
local anesthetics. In addition, the gene constructs can be
encapsulated within/administered in conjunction with
lipids/polycationic complexes.
[0361] Vaccines and immunogenic compositions can be sterilized and
if desired mixed with auxiliary agents, e.g., lubricants,
preservatives, stabilizers, wetting agents, emulsifiers, salts for
influencing osmotic pressure, buffers, coloring, flavoring and/or
aromatic substances and the like that do not deleteriously react
with the adjuvant or the administered nucleic acid or peptide.
[0362] The effective dose and method of administration of a
particular formulation can vary based on the individual patient and
the type and stage of the disease, as well as other factors known
to those of skill in the art. Therapeutic efficacy and toxicity can
be determined by standard pharmaceutical procedures in cell
cultures or experimental animals, e.g., ED50 (the dose
therapeutically effective in 50% of the population). The data
obtained from cell culture assays and animal studies can be used to
formulate a range of dosage for human use. The dosage lies
preferably within a range of circulating concentrations that
include the ED50 with no toxicity. The dosage varies within this
range depending upon the type of adjuvant derivative and antigen,
the dosage form employed, the sensitivity of the patient, and the
route of administration.
[0363] Since many adjuvants (e.g., ribavirin) have been on the
market for several years, many dosage forms and routes of
administration are known. All known dosage forms and routes of
administration can be provided within the context of the
embodiments described herein. Preferably, an amount of adjuvant
that is effective to enhance an immune response to an antigen in an
animal can be considered to be an amount that is sufficient to
achieve a blood serum level of antigen approximately 0.25-12.5
.mu.g/ml in the animal, preferably, about 2.5 .mu.g/ml. In some
embodiments, the amount of adjuvant is determined according to the
body weight of the animal to be given the vaccine. Accordingly, the
amount of adjuvant in a vaccine formulation can be from about
0.1-6.0 mg/kg body weight. That is, some embodiments have an amount
of adjuvant that corresponds to approximately 0.1-1.0 mg/kg,
1.1-2.0 mg/kg, 2.1-3.0 mg/kg, 3.1-4.0 mg/kg, 4.1-5.0 mg/kg, and
5.1-6.0 mg/kg body weight of an animal. More conventionally, the
vaccines contain approximately 0.25 mg-2000 mg of adjuvant. That
is, some embodiments have approximately 250 .mu.g, 500 .mu.g, 1 mg,
25 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400
mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg,
850 mg, 900 mg, Ig, 1.1 g, 1.2 g, 1.3 g, 1.4 g, 1.5 g, 1.6 g, 1.7
g, 1.8 g, 1.9 g, and 2 g of adjuvant.
[0364] As one of skill in the art will appreciate, the amount of
antigens in a vaccine can vary depending on the type of antigen and
its immunogenicity. The amount of antigens in the vaccine can vary
accordingly. Nevertheless, as a general guide, the vaccines can
have approximately 1 .mu.g, 5 .mu.g, 1 .mu.g, 20 .mu.g, 40 .mu.g,
80 .mu.g, 100 .mu.g, 0.25 mg-5 mg, 5-10 mg, 10-100 mg, 100-500 mg,
and upwards of 2000 mg of an antigen described herein, for example.
Preferably, the amount of antigen is 0.1 .mu.g-1 mg, desirably, 0.1
.mu.g-100 .mu.g, preferably 3 .mu.g-50 .mu.g, and, most preferably,
7 .mu.g, 8 .mu.g, 9 .mu.g, 10 .mu.g, 11 .mu.g-20 .mu.g, when said
antigen is a nucleic acid.
[0365] In some approaches described herein, the exact amount of
adjuvant and/or antigen is chosen by the individual physician in
view of the patient to be treated. Further, the amounts of adjuvant
can be added in combination to or separately from the same or
equivalent amount of antigen and these amounts can be adjusted
during a particular vaccination protocol so as to provide
sufficient levels in light of patient-specific or antigen-specific
considerations. In this vein, patient-specific and antigen-specific
factors that can be taken into account include, but are not limited
to, the severity of the disease state of the patient, age, and
weight of the patient, diet, time and frequency of administration,
drug combination(s), reaction sensitivities, and tolerance/response
to therapy.
Ribavirin
[0366] Nucleoside analogs have been widely used in anti-viral
therapies due to their capacity to reduce viral replication.
(Hosoya et al., J. Inf. Dis., 168:641-646 (1993)). ribavirin
(1-.beta.-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) is a
synthetic guanosine analog that has been used to inhibit RNA and
DNA virus replication. (Huffman et al., Antimicrob. Agents.
Chemother., 3:235 (1973); Sidwell et al., Science, 177:705 (1972)).
Ribavirin has been shown to be a competitive inhibitor of inositol
mono-phosphate (IMP) dehydrogenase (IMPDH), which converts IMP to
IMX (which is then converted to GMP). De Clercq, Anti viral Agents:
characteristic activity spectrum depending on the molecular target
with which they interact, Academic press, Inc., New York N.Y., pp.
1-55 (1993). Intracellular pools of GTP become depleted as a result
of long term ribavirin treatment.
[0367] In addition to antiviral activity, investigators have
observed that some guanosine analogs have an effect on the immune
system. (U.S. Pat. Nos. 6,063,772 and 4,950,647). Ribavirin has
been shown to inhibit functional humoral immune responses (Peavy et
al., J. Immunol., 126:861-864 (1981); Powers et al., Antimicrob.
Agents. Chemother., 22:108-114 (1982)) and IgE-mediated modulation
of mast cell secretion. (Marquardt et al., J. Pharmacol. Exp.
Therapeutics, 240:145-149 (1987)). Some investigators report that a
daily oral therapy of ribavirin has an immune modulating effect on
humans and mice. (Hultgren et al., J. Gen. Virol., 79:2381-2391
(1998) and Cramp et al., Gastron. Enterol., 118:346-355 (2000)).
Nevertheless, the current understanding of the effects of ribavirin
on the immune system is in its infancy. As disclosed below,
ribavirin was found to be a potent adjuvant.
Example 9
[0368] In a first set of experiments, groups of three to five
Balb/c mice (BK Universal, Uppsala, Sweden) were immunized i.p or
s.c. (e.g., at the base of the tail) with 10 .mu.g or 100 .mu.g of
recombinant hepatitis C virus non-structural 3 (rNS3) protein. The
rNS3 was dissolved in phosphate buffered saline (PBS) alone or PBS
containing 1 mg ribavirin (obtained from ICN, Costa Mesa, Calif.).
Mice were injected with a total volume of 100 .mu.l per
injection.
[0369] At two and four weeks following i.p. immunization, all mice
were bled by retro-orbital sampling. Serum samples were collected
and analyzed for the presence of antibodies to rNS3. To determine
the antibody titer, an enzyme immunoassay (EIA) was performed. (See
e.g., Hultgren et al., J Gen Virol. 79:2381-91 (1998) and Hultgren
et al., Clin. Diagn. Lab. Immunol. 4:630-632 (1997)). The antibody
levels were recorded as the highest serum dilution giving an
optical density at 405 nm more than twice that of non-immunized
mice.
[0370] Mice that received 10 .mu.g or 100 .mu.g rNS3 mixed with 1
mg ribavirin in PBS displayed consistently higher levels of NS3
antibodies. The antibody titer that was detected by EIA at two
weeks post-immunization is shown in FIG. 14. The vaccine
formulations having 1 mg of ribavirin and either 10 .mu.g or 100
.mu.g of rNS3 induced a significantly greater antibody titer than
the vaccine formulations composed of only rNS3.
[0371] In a second set of experiments, groups of eight Balb/c mice
were immunized intraperitoneally with 10 or 50 .mu.g of rNS3 in 100
.mu.l phosphate buffered saline containing either 0 mg, 1 mg, 3 mg,
or 10 mg ribavirin (Sigma). At four, six and eight weeks the mice
were bled and serum was separated and frozen. After completion of
the study, sera were tested for the levels of antibodies to
recombinant NS3, as described above. Mean antibody levels to rNS3
were compared between the groups using Student's t-test (parametric
analysis) or Mann-Whitney (non-parametric analysis) and the
software package StatView 4.5 (Abacus Concepts, Berkely, Calif.).
The adjuvant effect of ribavirin when added in three doses to 10
.mu.g of rNS3 are provided in TABLE 14. The adjuvant effect of
ribavirin when added in three doses to 50 .mu.g of rNS3 are
provided in TABLE 15. Parametrical comparison of the mean rNS3
antibody titres in mice receiving different 10 .mu.g or 50 .mu.g of
rNS3 and different doses of ribavirin are provided in TABLES 15 and
16, respectively. Non-parametrical comparison of mean NS3 antibody
titres in mice receiving different 10 .mu.g or 50 .mu.g of rNS3 and
different doses of ribavirin are provided in TABLES 17-19,
respectively. The values given represent end point titres to
recombinant rNS3.
TABLE-US-00015 TABLE 14 Amount Amount Antibody titre ribavirin
immunogen to rNS3 at indicated week (mg/dose) (.mu.g/dose) Mouse ID
Week 4 Week 6 Week 8 None 10 5:1 300 1500 1500 None 10 5:2 <60
7500 1500 None 10 5:3 <60 1500 300 None 10 5:4 60 1500 1500 None
10 5:5 <60 1500 nt None 10 5:6 60 1500 1500 None 10 5:7 <60
7500 7500 None 10 5:8 300 37500 7500 Group mean titre (mean .+-.
SD) 180 .+-. 139 7500 .+-. 12421 3042 .+-. 3076 1 10 6:1 300 37500
37500 1 10 6:2 <60 1500 1500 1 10 6:3 300 37500 187500 1 10 6:4
300 37500 7500 1 10 6:5 60 nt nt 1 10 6:6 <60 37500 7500 1 10
6:7 <60 37500 7500 1 10 6:8 300 7500 7500 Group mean titre (mean
.+-. SD) 252 .+-. 107 28071 .+-. 16195 36642 .+-. 67565 3 10 7:1 60
37500 7500 3 10 7:2 60 37500 37500 3 10 7:3 300 7500 7500 3 10 7:4
300 37500 7500 3 10 7:5 300 37500 37500 3 10 7:6 300 37500 37500 3
10 7:7 60 7500 7500 3 10 7:8 60 37500 37500 Group mean titre (mean
.+-. SD) 180 .+-. 128 30000 .+-. 13887 22500 .+-. 34637 10 10 8:1
300 37500 37500 10 10 8:2 300 37500 37500 10 10 8:3 <60 300 300
10 10 8:4 60 7500 7500 10 10 8:5 <60 300 300 10 10 8:6 <60
37500 37500 10 10 8:7 <60 7500 7500 10 10 8:8 <60 nt nt Group
mean titre (mean .+-. SD) 220 .+-. 139 18300 .+-. 18199 18300 .+-.
18199
TABLE-US-00016 TABLE 15 Amount Amount Antibody titre to rNS3 at
ribavirin immunogen indicated week (mg/dose) (.mu.g/dose) Mouse ID
Week 4 Week 6 Week 8 None 50 1:1 60 7500 7500 None 50 1:2 60 7500
7500 None 50 1:3 60 7500 7500 None 50 1:4 <60 1500 300 None 50
1:5 300 37500 37500 None 50 1:6 60 7500 7500 None 50 1:7 60 37500
7500 None 50 1:8 -- -- -- Group mean titre (mean .+-. SD) 100 .+-.
98 15214 .+-. 15380 10757 .+-. 12094 1 50 2:1 60 7500 7500 1 50 2:2
300 37500 7500 1 50 2:3 60 187500 7500 1 50 2:4 60 37500 187500 1
50 2:5 60 37500 7500 1 50 2:6 60 37500 37500 1 50 2:7 300 37500
7500 1 50 2:8 300 37500 37500 Group mean titre (mean .+-. SD) 150
.+-. 124 52500 .+-. 55549 37500 .+-. 62105 3 50 3:1 60 37500 7500 3
50 3:2 300 37500 37500 3 50 3:3 300 37500 7500 3 50 3:4 60 37500
7500 3 50 3:5 300 37500 7500 3 50 3:6 60 37500 7500 3 50 3:7 --
7500 37500 3 50 3:8 1500 7500 37500 Group mean titre (mean .+-. SD)
387 .+-. 513 30000 .+-. 13887 18750 .+-. 15526 10 50 4:1 300 7500
7500 10 50 4:2 300 37500 37500 10 50 4:3 60 7500 7500 10 50 4:4 60
7500 7500 10 50 4:5 60 1500 1500 10 50 4:6 60 7500 37500 10 50 4:7
-- 7500 7500 10 50 8:8 60 37500 7500 Group mean titre (mean .+-.
SD) 140 .+-. 124 10929 .+-. 11928 15214 .+-. 15380
TABLE-US-00017 TABLE 16 Group Week Mean .+-. SD Group Mean .+-. SD
analysis p-value 10 .mu.g NS3/ 4 180 .+-. 139 10 .mu.g NS3/ 252
.+-. 107 Students 0.4071 no ribavirin 1 mg ribavirin t-test 6 7500
.+-. 12421 28071 .+-. 16195 Students 0.0156* t-test 8 3042 .+-.
3076 36642 .+-. 67565 Students 0.2133 t-test 10 .mu.g NS3/ 4 180
.+-. 139 10 .mu.g NS3/ 180 .+-. 128 Students 1.000 no ribavirin 3
mg ribavirin t-test 6 7500 .+-. 12421 30000 .+-. 13887 Students
0.0042** t-test 8 3042 .+-. 3076 22500 .+-. 34637 Students 0.0077**
t-test 10 .mu.g NS3/ 4 180 .+-. 139 10 .mu.g NS3/ 220 .+-. 139
Students 0.7210 no ribavirin 10 mg ribavirin t-test 6 7500 .+-.
12421 18300 .+-. 18199 Students 0.1974 t-test 8 3042 .+-. 3076
18300 .+-. 18199 Students 0.0493* t-test
TABLE-US-00018 TABLE 17 Group Week Mean .+-. SD Group Mean .+-. SD
analysis p-value 50 .mu.g NS3/ 4 100 .+-. 98 50 .mu.g NS3/ 150 .+-.
124 Students 0.4326 no ribavirin 1 mg ribavirin t-test 6 15214 .+-.
15380 52500 .+-. 55549 Students 0.1106 t-test 8 10757 .+-. 12094
37500 .+-. 62105 Students 0.2847 t-test 50 .mu.g NS3/ 4 100 .+-. 98
50 .mu.g NS3/ 387 .+-. 513 Students 0.2355 no ribavirin 3 mg
ribavirin t-test 6 15214 .+-. 15380 30000 .+-. 13887 Students
0.0721 t-test 8 10757 .+-. 12094 18750 .+-. 15526 Students 0.2915
t-test 50 .mu.g NS3/ 4 100 .+-. 98 50 .mu.g NS3/ 140 .+-. 124
Students 0.5490 no ribavirin 10 mg ribavirin t-test 6 15214 .+-.
15380 10929 .+-. 11928 Students 0.5710 t-test 8 10757 .+-. 12094
15214 .+-. 15380 Students 0.5579 t-test Significance levels: NS =
not significant; * = p < 0.05; ** = p < 0.01; *** = p <
0.001
TABLE-US-00019 TABLE 18 Group Week Mean .+-. SD Group Mean .+-. SD
analysis p-value 10 .mu.g NS3/ 4 180 .+-. 139 10 .mu.g NS3/ 252
.+-. 107 Mann- 0.4280 no ribavirin 1 mg ribavirin Whitney 6 7500
.+-. 12421 28071 .+-. 16195 Mann- 0.0253* Whitney 8 3042 .+-. 3076
36642 .+-. 67565 Mann- 0.0245* Whitney 10 .mu.g NS3/ 4 180 .+-. 139
10 .mu.g NS3/ 180 .+-. 128 Mann- 0.0736 no ribavirin 3 mg ribavirin
Whitney 6 7500 .+-. 12421 30000 .+-. 13887 Mann- 0.0050** Whitney 8
3042 .+-. 3076 22500 .+-. 34637 Mann- 0.0034** Whitney 10 .mu.g
NS3/ 4 180 .+-. 139 10 .mu.g NS3/ 220 .+-. 139 Mann- 0.8986 no
ribavirin 10 mg ribavirin Whitney 6 7500 .+-. 12421 18300 .+-.
18199 Mann- 0.4346 Whitney 8 3042 .+-. 3076 18300 .+-. 18199 Mann-
0.2102 Whitney
TABLE-US-00020 TABLE 19 Group Week Mean .+-. SD Group Mean .+-. SD
analysis p-value 50 .mu.g NS3/ 4 100 .+-. 98 50 .mu.g NS3/ 150 .+-.
124 Mann- 0.1128 no ribavirin 1 mg ribavirin Whitney 6 15214 .+-.
15380 52500 .+-. 55549 Mann- 0.0210* Whitney 8 10757 .+-. 12094
37500 .+-. 62105 Mann- 0.1883 Whitney 50 .mu.g NS3/ 4 100 .+-. 98
50 .mu.g NS3/ 387 .+-. 513 Mann- 0.1400 no ribavirin 3 mg ribavirin
Whitney 6 15214 .+-. 15380 30000 .+-. 13887 Mann- 0.0679 Whitney 8
10757 .+-. 12094 18750 .+-. 15526 Mann- 0.2091 Whitney 50 .mu.g
NS3/ 4 100 .+-. 98 50 .mu.g NS3/ 140 .+-. 124 Mann- 0.4292 no
ribavirin 10 mg ribavirin Whitney 6 15214 .+-. 15380 10929 .+-.
11928 Mann- 0.9473 Whitney 8 10757 .+-. 12094 15214 .+-. 15380
Mann- 0.6279 Whitney Significance levels: NS = not significant; *=
p < 0.05; ** = p < 0.01; *** = p < 0.001
[0372] The data above demonstrates that ribavirin facilitates or
enhances an immune response to an HCV antigen or HCV epitopes. A
potent immune response to rNS3 was elicited after immunization with
a vaccine composition comprising as little as 1 mg ribavirin and 10
.mu.g of rNS3 antigen. The data above also provide evidence that
the amount of ribavirin that is sufficient to facilitate an immune
response to an antigen is between 1 and 3 mg per injection for a
25-30 g Balb/c mouse. It should be realized, however, that these
amounts are intended for guidance only and should not be
interpreted to limit the scope of the invention in any way.
Nevertheless, the data shows that vaccine compositions comprising
approximately 1 to 3 mg doses of ribavirin induce an immune
response that is more than 12 times higher than the immune response
elicited in the absence of without ribavirin. Thus, ribavirin has a
significant adjuvant effect on the humoral immune response of an
animal and thereby, enhances or facilitates the immune response to
the antigen. The example below describes experiments that were
performed to better understand the amount of ribavirin needed to
enhance or facilitate an immune response to an antigen.
Example 10
[0373] To determine a dose of ribavirin that is sufficient to
provide an adjuvant effect, the following experiments were
performed. In a first set of experiments, groups of mice (three per
group) were immunized with a 20 .mu.g rNS3 alone or a mixture of 20
.mu.g rNS3 and 0.1 mg, 1 mg, or 10 mg ribavirin. The levels of
antibody to the antigen were then determined by EIA. The mean
endpoint titers at weeks 1 and 3 were plotted and are shown in FIG.
15. It was discovered that the adjuvant effect provided by
ribavirin had different kinetics depending on the dose of ribavirin
provided. For example, even low doses (<1 mg) of ribavirin were
found to enhance antibody levels at week one but not at week three,
whereas, higher doses (1-10 mg) were found to enhance antibody
levels at week three.
[0374] A second set of experiments was also performed. In these
experiments, groups of mice were injected with vaccine compositions
comprising various amounts of ribavirin and rNS3 and the IgG
response in these animals was monitored. The vaccine compositions
comprised approximately 100 .mu.l phosphate buffered saline and 20
.mu.g rNS3 with or without 0.1 mg, 1.0 mg, or 10 mg ribavirin
(Sigma). The mice were bled at week six and rNS3-specific IgG
levels were determined by EIA as described previously. As shown in
TABLE 20, the adjuvant effects on the sustained antibody levels
were most obvious in the dose range of 1 to 10 mg per injection for
a 25-30 g mouse.
TABLE-US-00021 TABLE 20 Amount (mg) ribavirin mixed with the
Endpoint titre of rNS3 IgG at indicated week Immunogen immunogen
Mouse ID Week 1 Week 2 Week 3 20 .mu.g rNS3 None 1 60 360 360 20
.mu.g rNS3 None 2 360 360 2160 20 .mu.g rNS3 None 3 360 2160 2160
Mean 260 .+-. 173 960 .+-. 1039 1560 .+-. 1039 20 .mu.g rNS3 0.1 4
2160 12960 2160 20 .mu.g rNS3 0.1 5 60 60 60 20 .mu.g rNS3 0.1 6
<60 2160 2160 1110 .+-. 1484 5060 .+-. 6921 1460 .+-. 1212 20
.mu.g rNS3 1.0 7 <60 60 12960 20 .mu.g rNS3 1.0 8 <60 2160
2160 20 .mu.g rNS3 1.0 9 360 2160 2160 Mean 360 1460 .+-. 1212 5760
.+-. 6235 20 .mu.g rNS3 10.0 10 360 12960 77760 20 .mu.g rNS3 10.0
11 <60 2160 12960 20 .mu.g rNS3 10.0 12 360 2160 2160 Mean 360
5760 .+-. 6235 30960 .+-. 40888
[0375] In a third set of experiments, the adjuvant effect of
ribavirin after primary and booster injections was investigated. In
these experiments, mice were given two intraperitoneal injections
of a vaccine composition comprising 10 .mu.g rNS3 with or without
ribavirin and the IgG subclass responses to the antigen was
monitored, as before. Accordingly, mice were immunized with 100
.mu.l phosphate buffered containing 10 .mu.g recombinant NS3 alone,
with or without 0.1 or 1.0 mg ribavirin (Sigma) at weeks 0 and 4.
The mice were bled at week six and NS3-specific IgG subclasses were
determined by EIA as described previously. As shown in TABLE 21,
the addition of ribavirin to the immunogen prior to the injection
does not change the IgG subclass response in the NS3-specific
immune response. Thus, the adjuvant effect of a vaccine composition
comprising ribavirin and an antigen can not be explained by a shift
in of the Th1/Th2-balance. It appears that another mechanism may be
responsible for the adjuvant effect of ribavirin.
TABLE-US-00022 TABLE 21 Amount (mg) ribavirin mixed Mouse Endpoint
titre of indicated NS3 IgG subclass Immunogen with the immunogen ID
IgG1 IgG2a IgG2b IgG3 10 .mu.g rNS3 None 1 360 60 <60 60 10
.mu.g rNS3 None 2 360 <60 <60 60 10 .mu.g rNS3 None 3 2160 60
<60 360 Mean 960 .+-. 1039 60 -- 160 .+-. 173 10 .mu.g rNS3 0.1
4 360 <60 <60 60 10 .mu.g rNS3 0.1 5 60 <60 <60 <60
10 .mu.g rNS3 0.1 6 2160 60 60 360 860 .+-. 1136 60 60 210 .+-. 212
10 .mu.g rNS3 1.0 7 2160 <60 <60 60 10 .mu.g rNS3 1.0 8 360
<60 <60 <60 10 .mu.g rNS3 1.0 9 2160 <60 <60 60 Mean
1560 .+-. 1039 -- -- 60
[0376] The data presented in this example further verify that
ribavirin can be administered as an adjuvant and establish that
that the dose of ribavirin can modulate the kinetics of the
adjuvant effect. The example below describes another assay that was
performed to evaluate the ability of ribavirin to enhance or
facilitate an immune response to an antigen.
Example 11
[0377] This assay can be used with any ribavirin derivative or
combinations of ribavirin derivatives to determine the extent that
a particular vaccine formulation modulates a cellular immune
response. To determine CD4.sup.+ T cell responses to a
ribavirin-containing vaccine, groups of mice were immunized s.c.
with either 100 .mu.g rNS3 in PBS or 100 .mu.g rNS3 and 1 mg
ribavirin in PBS. The mice were sacrificed ten days
post-immunization and their lymph nodes were harvested and drained.
In vitro recall assays were then performed. (See e.g., Hultgren et
al., J Gen Virol. 79:2381-91 (1998) and Hultgren et al., Clin.
Diagn. Lab. Immunol. 4:630-632 (1997)). The amount of CD4.sup.+ T
cell proliferation was determined at 96 h of culture by the
incorporation of [.sup.3H] thymidine.
[0378] As shown in FIG. 16, mice that were immunized with 100 .mu.g
rNS3 mixed with 1 mg ribavirin had a much greater T cell
proliferative response than mice that were immunized with 100 .mu.g
rNS3 in PBS. This data provides more evidence that ribavirin
enhances or facilitates a cellular immune response (e.g., by
promoting the effective priming of T cells).
[0379] Additional experiments were conducted to verify that
ribavirin enhances the immune response to commercially available
vaccine preparations. The example below describes the use of
ribavirin in conjunction with a commercial HBV vaccine
preparation.
Example 12
[0380] The adjuvant effect of ribavirin was tested when mixed with
two doses of a commercially available vaccine containing HBsAg and
alum. (Engerix, SKB). Approximately 0.2 .mu.g or 2 .mu.g of Engerix
vaccine was mixed with either PBS or 1 mg ribavirin in PBS and the
mixtures were injected intra peritoneally into groups of mice
(three per group). A booster containing the same mixture was given
on week four and all mice were bled on week six. The serum samples
were diluted from 1:60 to 1:37500 and the dilutions were tested by
EIA, as described above, except that purified human HBsAg was used
as the solid phase antigen. As shown in TABLE 22, vaccine
formulations having ribavirin enhanced the response to 2 .mu.g of
an existing vaccine despite the fact that the vaccine already
contained alum. That is, by adding ribavirin to a suboptimal
vaccine dose (i.e., one that does not induce detectable antibodies
alone) antibodies became detectable, providing evidence that the
addition of ribavirin allows for the use of lower antigen amounts
in a vaccine formulation without compromising the immune
response.
TABLE-US-00023 TABLE 22 End point antibody titer to HBsAg in EIA
0.02 .mu.g Engerix 0.2 .mu.g Engerix No ribavirin 1 mg ribavirin No
ribavirin 1 mg ribavirin Week #1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3 6
<60 <60 <60 <60 <60 <60 <60 <60 <60 300
60 <60
[0381] The ribavirin used in the experiments above was obtained
from commercial suppliers (e.g., Sigma and ICN). The ribavirin that
can be used with the embodiments described herein can also be
obtained from commercial suppliers or can be synthesized. The
ribavirin and/or the antigen can be formulated with and without
modification. For example, the ribavirin can be modified or
derivatized to make a more stable molecule and/or a more potent
adjuvant. By one approach, the stability of ribavirin can be
enhanced by coupling the molecules to a support such as a
hydrophilic polymer (e.g., polyethylene glycol).
[0382] Many more ribavirin derivatives can be generated using
conventional techniques in rational drug design and combinatorial
chemistry. For example, Molecular Simulations Inc. (MSI), as well
as many other suppliers, provide software that allows one of skill
to build a combinatorial library of organic molecules. The
C2.Analog Builder program, for example, can be integrated with
MSI's suite of Cerius2 molecular diversity software to develop a
library of ribavirin derivatives that can be used with the
embodiments described herein. (See e.g.,
http://msi.com/life/products/cerius2/index.html).
[0383] By one approach, the chemical structure of ribavirin is
recorded on a computer readable media and is accessed by one or
more modeling software application programs. The C2.Analog Builder
program in conjunction with C2Diversity program allows the user to
generate a very large virtual library based on the diversity of
R-groups for each substituent position, for example. Compounds
having the same structure as the modeled ribavirin derivatives
created in the virtual library are then made using conventional
chemistry or can be obtained from a commercial source.
[0384] The newly manufactured ribavirin derivatives can then be
screened in assays, which determine the extent of adjuvant activity
of the molecule and/or the extent of its ability to modulate of an
immune response. Some assays may involve virtual drug screening
software, such as C2.Ludi. C2.Ludi is a software program that
allows a user to explore databases of molecules (e.g., ribavirin
derivatives) for their ability to interact with the active site of
a protein of interest (e.g., RAC2 or another GTP binding protein).
Based upon predicted interactions discovered with the virtual drug
screening software, the ribavirin derivatives can be prioritized
for further characterization in conventional assays that determine
adjuvant activity and/or the extent of a molecule to modulate an
immune response. The section below provides more explanation
concerning the methods of using the compositions described
herein.
Methods of Using the Vaccine Compositions and Immunogen
Preparations
[0385] Routes of administration of the embodiments described herein
include, but are not limited to, transdermal, parenteral,
gastrointestinal, transbronchial, and transalveolar. Transdermal
administration can be accomplished by application of a cream,
rinse, gel, etc. capable of allowing the adjuvant and HCV antigen
to penetrate the skin. Parenteral routes of administration include,
but are not limited to, electrical or direct injection such as
direct injection into a central venous line, intravenous,
intramuscular, intraperitoneal, intradermal, or subcutaneous
injection. Gastrointestinal routes of administration include, but
are not limited to, ingestion and rectal. Transbronchial and
transalveolar routes of administration include, but are not limited
to, inhalation, either via the mouth or intranasally.
[0386] Compositions having the adjuvant and HCV antigen that are
suitable for transdermal administration include, but are not
limited to, pharmaceutically acceptable suspensions, oils, creams,
and ointments applied directly to the skin or incorporated into a
protective carrier such as a transdermal device ("transdermal
patch"). Examples of suitable creams, ointments, etc. can be found,
for instance, in the Physician's Desk Reference. Examples of
suitable transdermal devices are described, for instance, in U.S.
Pat. No. 4,818,540 issued Apr. 4, 1989 to Chinen, et al.
[0387] Compositions having the adjuvant and HCV antigen that are
suitable for parenteral administration include, but are not limited
to, pharmaceutically acceptable sterile isotonic solutions. Such
solutions include, but are not limited to, saline, phosphate
buffered saline and oil preparations for injection into a central
venous line, intravenous, intramuscular, intraperitoneal,
intradermal, or subcutaneous injection.
[0388] Compositions having the adjuvant and HCV antigen that are
suitable for transbronchial and transalveolar administration
include, but not limited to, various types of aerosols for
inhalation. Devices suitable for transbronchial and transalveolar
administration of these are also embodiments. Such devices include,
but are not limited to, atomizers and vaporizers. Many forms of
currently available atomizers and vaporizers can be readily adapted
to deliver vaccines having ribavirin and an antigen.
[0389] Compositions having the adjuvant and HCV antigen that are
suitable for gastrointestinal administration include, but not
limited to, pharmaceutically acceptable powders, pills or liquids
for ingestion and suppositories for rectal administration.
[0390] The gene constructs described herein, in particular, may be
administered by means including, but not limited to, traditional
syringes, needleless injection devices, or "microprojectile
bombardment gene guns". Alternatively, the genetic vaccine may be
introduced by various means into cells that are removed from the
individual. Such means include, for example, ex vivo transfection,
electroporation, microinjection and microprojectile bombardment.
After the gene construct is taken up by the cells, they are
reimplanted into the individual. It is contemplated that otherwise
non-immunogenic cells that have gene constructs incorporated
therein can be implanted into the individual even if the vaccinated
cells were originally taken from another individual.
[0391] According to some embodiments, the gene construct is
administered to an individual using a needleless injection device.
According to some embodiments, the gene construct is simultaneously
administered to an individual intradermally, subcutaneously and
intramuscularly using a needleless injection device. Needleless
injection devices are well known and widely available. One having
ordinary skill in the art can, following the teachings herein, use
needleless injection devices to deliver genetic material to cells
of an individual. Needleless injection devices are well suited to
deliver genetic material to all tissue. They are particularly
useful to deliver genetic material to skin and muscle cells. In
some embodiments, a needleless injection device may be used to
propel a liquid that contains DNA molecules toward the surface of
the individual's skin. The liquid is propelled at a sufficient
velocity such that upon impact with the skin the liquid penetrates
the surface of the skin, permeates the skin and muscle tissue
therebeneath. Thus, the genetic material is simultaneously
administered intradermally, subcutaneously and intramuscularly. In
some embodiments, a needleless injection device may be used to
deliver genetic material to tissue of other organs in order to
introduce a nucleic acid molecule to cells of that organ.
[0392] Preferred embodiments concern methods of treating or
preventing HCV infection. In these embodiments, an animal in need
is provided an HCV antigen (e.g., a peptide antigen or nucleic
acid-based antigen, as described herein (SEQ. ID. NOs.: 1-27,
35-36, and 40-220 (including wild-type and codon optimized
sequences encoding SEQ ID NOs: 40-220) and an amount of adjuvant
sufficient to exhibit an adjuvant activity in said animal.
Accordingly, an animal can be identified as one in need by using
currently available diagnostic testing or clinical evaluation. The
adjuvant and antigen can be provided separately or in combination,
and other adjuvants (e.g., oil, alum, or other agents that enhance
an immune response) can also be provided to the animal in need.
[0393] Other embodiments of the invention include methods of
enhancing an immune response to an HCV antigen by providing an
animal in need with an amount of adjuvant (e.g., ribavirin) and one
or more of SEQ. ID. NOs.: 1-11, 35-36, and 40-220 (or a wild type
or codon-optimized nucleic acid encoding SEQ ID NOs: 40-220) or a
fragment thereof, preferably SEQ. ID. NOs.: 12-27 that is effective
to enhance said immune response. In these embodiments, an animal in
need of an enhanced immune response to an antigen is identified by
using currently available diagnostic testing or clinical
evaluation. By one approach, for example, an uninfected individual
is provided with the vaccine compositions described above in an
amount sufficient to elicit a cellular and humoral immune response
to NS3 so as to protect said individual from becoming infected with
HCV. In another embodiment, an HCV-infected individual is
identified and provided with a vaccine composition comprising
ribavirin and NS3 in an amount sufficient to enhance the cellular
and humoral immune response against NS3 so as to reduce or
eliminate the HCV infection. Such individual may be in the chronic
or acute phase of the infection. In yet another embodiment, an
HCV-infected individual suffering from HCC is provided with a
composition comprising an adjuvant and the NS3/4A fusion gene in an
amount sufficient to elicit a cellular and humoral immune response
against NS3-expressing tumor cells.
[0394] The next section describes some of the peptide embodiments
of the invention.
HCV Peptides
[0395] The embodied HCV peptides or derivatives thereof, include
but are not limited to, those containing as a primary amino acid
sequence all of the amino acid sequence substantially as depicted
in the Sequence Listing (SEQ. ID. NOs.: 2-11, 36, and SEQ ID NOs:
40-220) and fragments of SEQ. ID. NOs.: 2-11 and SEQ. ID. NO.: 36
that are at least four amino acids in length (e.g., SEQ. ID. NOs.:
14-16) including altered sequences in which functionally equivalent
amino acid residues are substituted for residues within the
sequence resulting in a silent change. Preferred fragments of a
sequence of SEQ. ID. NOs.: 2-11 and SEQ. ID. NO.: 36 are at least
four amino acids and comprise amino acid sequence unique to the
discovered NS3/4A peptide or mutants thereof including altered
sequences in which functionally equivalent amino acid residues are
substituted for residues within the sequence resulting in a silent
change. The HCV peptides can be, for example, at least 12-704 amino
acids in length (e.g., any number between 12-15, 15-20, 20-25,
25-50, 50-100, 100-150, 150-250, 250-500 or 500-704 amino acids in
length).
[0396] Embodiments also include HCV peptides that are substantially
identical to those described above. That is, HCV peptides that have
one or more amino acid residues within SEQ. ID. NOs.: 2-11, 36, and
40-220 and fragments thereof that are substituted by another amino
acid of a similar polarity that acts as a functional equivalent,
resulting in a silent alteration. Further, the HCV peptides can
have one or more amino acid residues fused to SEQ. ID. NOs.: 2-11,
36 and SEQ ID NO: 40-220 or a fragment thereof so long as the
fusion does not significantly alter the structure or function
(e.g., immunogenic properties) of the HCV peptide. Substitutes for
an amino acid within the sequence can be selected from other
members of the class to which the amino acid belongs. For example,
the non-polar (hydrophobic) amino acids include alanine, leucine,
isoleucine, valine, proline, phenylalanine, tryptophan, and
methionine. The polar neutral amino acids include glycine, serine,
threonine, cysteine, tyrosine, asparagine and glutamine. The
positively charged (basic) amino acids include arginine, lysine,
and histidine. The negatively charged (acidic) amino acids include
aspartic acid and glutamic acid. The aromatic amino acids include
phenylalanine, tryptophan, and tyrosine. Accordingly, the peptide
embodiments of the invention are said to be consisting essentially
of SEQ. ID. NOs.: 2-27, 36 and SEQ ID NOs: 40-220 in light of the
modifications described above.
[0397] The HCV peptides described herein can be prepared by
chemical synthesis methods (such as solid phase peptide synthesis)
using techniques known in the art such as those set forth by
Merrifield et al., J. Am. Chem. Soc. 85:2149 (1964), Houghten et
al., Proc. Natl. Acad. Sci. USA, 82:51:32 (1985), Stewart and Young
(Solid phase peptide synthesis, Pierce Chem Co., Rockford, Ill.
(1984), and Creighton, 1983, Proteins: Structures and Molecular
Principles, W. H. Freeman & Co., N.Y. Such polypeptides can be
synthesized with or without a methionine on the amino terminus.
Chemically synthesized HCV peptides can be oxidized using methods
set forth in these references to form disulfide bridges.
[0398] While the HCV peptides described herein can be chemically
synthesized, it can be more effective to produce these polypeptides
by recombinant DNA technology. Such methods can be used to
construct expression vectors containing the HCV nucleotide
sequences described above, for example, and appropriate
transcriptional and translational control signals. These methods
include, for example, in vitro recombinant DNA techniques,
synthetic techniques, and in vivo genetic recombination.
Alternatively, RNA capable of encoding an HCV nucleotide sequence
can be chemically synthesized using, for example, synthesizers.
See, for example, the techniques described in Oligonucleotide
Synthesis, 1984, Gait, M. J. ed., IRL Press, Oxford. Accordingly,
several embodiments concern cell lines that have been engineered to
express the embodied HCV peptides. For example, some cells are made
to express the HCV peptides of SEQ. ID. NOs.: 2-11, 36 and SEQ ID
NOs: 40-220 or fragments of these molecules (e.g., SEQ. ID. NOs.:
14-26).
[0399] A variety of host-expression vector systems can be utilized
to express the embodied HCV peptides. Suitable expression systems
include, but are not limited to, microorganisms such as bacteria
(e.g., E. coli or B. subtilis) transformed with recombinant
bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors
containing HCV nucleotide sequences; yeast (e.g., Saccharomyces,
Pichia) transformed with recombinant yeast expression vectors
containing the HCV nucleotide sequences; insect cell systems
infected with recombinant virus expression vectors (e.g.,
baculovirus) containing the HCV sequences; plant cell systems
infected with recombinant virus expression vectors (e.g.,
cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or
transformed with recombinant plasmid expression vectors (e.g., Ti
plasmid) containing HCV sequences; or mammalian cell systems (e.g.,
COS, CHO, BHK, 293, 3T3) harboring recombinant expression
constructs containing promoters derived from the genome of
mammalian cells (e.g., metallothionein promoter) or from mammalian
viruses (e.g., the adenovirus late promoter; the vaccinia virus
7.5K promoter).
[0400] In bacterial systems, a number of expression vectors can be
advantageously selected depending upon the use intended for the HCV
gene product being expressed. For example, when a large quantity of
such a protein is to be produced, for the generation of
pharmaceutical compositions of HCV peptide or for raising
antibodies to the HCV peptide, for example, vectors which direct
the expression of high levels of fusion protein products that are
readily purified can be desirable. Such vectors include, but are
not limited, to the E. coli expression vector pUR278 (Ruther et
al., EMBO J., 2:1791 (1983), in which the HCV coding sequence can
be ligated individually into the vector in frame with the lacZ
coding region so that a fusion protein is produced; pIN vectors
(Inouye & Inouye, Nucleic Acids Res., 13:3101-3109 (1985); Van
Heeke & Schuster, J. Biol. Chem., 264:5503-5509 (1989)); and
the like. The pGEX vectors can also be used to express foreign
polypeptides as fusion proteins with glutathione S-transferase
(GST). In general, such fusion proteins are soluble and can be
purified from lysed cells by adsorption to glutathione-agarose
beads followed by elution in the presence of free glutathione. The
PGEX vectors are designed to include thrombin or factor Xa protease
cleavage sites so that the cloned target gene product can be
released from the GST moiety.
[0401] In an insect system, Autographa californica nuclear
polyhedrosis virus (AcNPV) is used as a vector to express foreign
genes. The virus grows in Spodoptera frugiperda cells. The HCV
coding sequence can be cloned individually into non-essential
regions (for example the polyhedrin gene) of the virus and placed
under control of an AcNPV promoter (for example the polyhedrin
promoter). Successful insertion of an HCV gene coding sequence will
result in inactivation of the polyhedrin gene and production of
non-occluded recombinant virus, (i.e., virus lacking the
proteinaceous coat coded for by the polyhedrin gene). These
recombinant viruses are then used to infect Spodoptera frugiperda
cells in which the inserted gene is expressed. (See e.g., Smith et
al., J. Virol. 46: 584 (1983); and Smith, U.S. Pat. No.
4,215,051).
[0402] In mammalian host cells, a number of viral-based expression
systems can be utilized. In cases where an adenovirus is used as an
expression vector, the HCV nucleotide sequence of interest can be
ligated to an adenovirus transcription/translation control complex,
e.g., the late promoter and tripartite leader sequence. This
chimeric gene can then be inserted in the adenovirus genome by in
vitro or in vivo recombination. Insertion in a non-essential region
of the viral genome (e.g., region E1 or E3) will result in a
recombinant virus that is viable and capable of expressing the HCV
gene product in infected hosts. (See e.g., Logan & Shenk, Proc.
Natl. Acad. Sci. USA 81:3655-3659 (1984)). Specific initiation
signals can also be required for efficient translation of inserted
HCV nucleotide sequences. These signals include the ATG initiation
codon and adjacent sequences.
[0403] However, in cases where only a portion of the HCV coding
sequence is inserted, exogenous translational control signals,
including, perhaps, the ATG initiation codon, can be provided.
Furthermore, the initiation codon can be in phase with the reading
frame of the desired coding sequence to ensure translation of the
entire insert. These exogenous translational control signals and
initiation codons can be of a variety of origins, both natural and
synthetic. The efficiency of expression can be enhanced by the
inclusion of appropriate transcription enhancer elements,
transcription terminators, etc. (See Bittner et al., Methods in
Enzymol., 153:516-544 (1987)).
[0404] In addition, a host cell strain can be chosen, which
modulates the expression of the inserted sequences, or modifies and
processes the gene product in the specific fashion desired. Such
modifications (e.g., glycosylation) and processing (e.g., cleavage)
of protein products are important for the function of the protein.
Different host cells have characteristic and specific mechanisms
for the post-translational processing and modification of proteins
and gene products. Appropriate cell lines or host systems can be
chosen to ensure the correct modification and processing of the
foreign protein expressed. To this end, eukaryotic host cells that
possess the cellular machinery for proper processing of the primary
transcript, glycosylation, and phosphorylation of the gene product
can be used. Such mammalian host cells include, but are not limited
to, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, and WI38.
[0405] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
that stably express the HCV peptides described above can be
engineered. Rather than using expression vectors that contain viral
origins of replication, host cells can be transformed with DNA
controlled by appropriate expression control elements (e.g.,
promoter, enhancer sequences, transcription terminators,
polyadenylation sites, etc.), and a selectable marker. Following
the introduction of the foreign DNA, engineered cells are allowed
to grow for 1-2 days in an enriched media, and then are switched to
a selective media. The selectable marker in the recombinant plasmid
confers resistance to the selection and allows cells to stably
integrate the plasmid into their chromosomes and grow to form foci
which in turn are cloned and expanded into cell lines. This method
is advantageously used to engineer cell lines which express the HCV
gene product.
[0406] A number of selection systems can be used, including but not
limited to the herpes simplex virus thymidine kinase (Wigler, et
al., Cell 11:223 (1977), hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl.
Acad. Sci. USA 48:2026 (1962)), and adenine
phosphoribosyltransferase (Lowy, et al., Cell 22:817 (1980)) genes
can be employed in tk.sup.-, hgprt.sup.- or aprt.sup.- cells,
respectively. Also, antimetabolite resistance can be used as the
basis of selection for the following genes: dhfr, which confers
resistance to methotrexate (Wigler, et al., Proc. Natl. Acad. Sci.
USA 77:3567 (1980); O'Hare, et al., Proc. Natl. Acad. Sci. USA
78:1527 (1981)); gpt, which confers resistance to mycophenolic acid
(Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072 (1981));
neo, which confers resistance to the aminoglycoside G-418
(Colberre-Garapin, et al., J. Mol. Biol. 150:1 (1981)); and hygro,
which confers resistance to hygromycin (Santerre, et al., Gene
30:147 (1984)).
[0407] Alternatively, any fusion protein can be readily purified by
utilizing an antibody specific for the fusion protein being
expressed. For example, a system described by Janknecht et al.
allows for the ready purification of non-denatured fusion proteins
expressed in human cell lines. (Janknecht, et al., Proc. Natl.
Acad. Sci. USA 88: 8972-8976 (1991)). In this system, the gene of
interest is subcloned into a vaccinia recombination plasmid such
that the gene's open reading frame is translationally fused to an
amino-terminal tag consisting of six histidine residues. Extracts
from cells infected with recombinant vaccinia virus are loaded onto
Ni.sup.2+ nitriloacetic acid-agarose columns and histidine-tagged
proteins are selectively eluted with imidazole-containing buffers.
The example below describes a method that was used to express the
HCV peptides encoded by the embodied nucleic acids.
Example 13
[0408] To characterize NS3/4A-pVAX, MSLF1-pVAX, and the NS3/4A
mutant constructs, described in Example 1, the plasmids were
transcribed and translated in vitro, and the resulting polypeptides
were visualized by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE). In vitro transcription and translation
were performed using the T7 coupled reticulocyte lysate system
(Promega, Madison, Wis.) according to the manufacturer's
instructions. All in vitro translation reactions of the expression
constructs were carried out at 30.degree. C. with .sup.35S-labeled
methionine (Amersham International, Plc, Buckinghamshire, UK). The
labeled proteins were separated by 12% SDS-PAGE and visualized by
exposure to X-ray film (Hyper Film-MP, Amersham) for 6-18
hours.
[0409] The in vitro analysis revealed that all proteins were
expressed to high amounts from their respective expression
constructs. The rNS3 construct (NS3-pVAX vector) produced a single
peptide of approximately 61 kDa, whereas, the mutant constructs
(e.g., the TGT construct (NS3/4A-TGT-pVAX) and the RGT construct
(NS3/4A-RGT-pVAX)) produced a single polypeptide of approximately
67 kDa, which is identical to the molecular weight of the uncleaved
NS3/4A peptide produced from the NS3/4A-pVAX construct. The cleaved
product produced from the expressed NS3/4A peptide was
approximately 61 kDa, which was identical in size to the rNS3
produced from the NS3-pVAX vector. These results demonstrated that
the expression constructs were functional, the NS3/4A construct was
enzymatically active, the rNS3 produced a peptide of the predicted
size, and the breakpoint mutations completely abolished cleavage at
the NS3-NS4A junction.
[0410] To compare the translation efficiency from the NS3/4A-pVAX
and MSLF1-pVAX plasmids, the amount of input DNA was serially
diluted prior to addition to the assay. Serial dilutions of the
plasmids revealed that the MSLF1 plasmid gave stronger bands at
higher dilutions of the plasmid than the wild-type NS3/4A plasmid,
providing evidence that in vitro transcription and translation was
more efficient from the MSLF1 plasmid. The NS3/4A-pVAX and MSLF1
plasmids were then analyzed for protein expression using
transiently transfected Hep-G2 cells. Similar results were obtained
in that the MSLF-1 gene provided more efficient expression of NS3
than the native NS3/4A gene.
[0411] The sequences, constructs, vectors, clones, and other
materials comprising the embodied HCV nucleic acids and peptides
can be in enriched or isolated form. As used herein, "enriched"
means that the concentration of the material is many times its
natural concentration, for example, at least about 2, 5, 10, 100,
or 1000 times its natural concentration, advantageously 0.01%, by
weight, preferably at least about 0.1% by weight. Enriched
preparations from about 0.5% or more, for example, 1%, 5%, 10%, and
20% by weight are also contemplated. The term "isolated" requires
that the material be removed from its original environment (e.g.,
the natural environment if it is naturally occurring). For example,
a naturally-occurring polynucleotide present in a living animal is
not isolated, but the same polynucleotide, separated from some or
all of the coexisting materials in the natural system, is isolated.
It is also advantageous that the sequences be in purified form. The
term "purified" does not require absolute purity; rather, it is
intended as a relative definition. Isolated proteins have been
conventionally purified to electrophoretic homogeneity by Coomassie
staining, for example. Purification of starting material or natural
material to at least one order of magnitude, preferably two or
three orders, and more preferably four or five orders of magnitude
is expressly contemplated.
[0412] The HCV gene products described herein can also be expressed
in plants, insects, and animals so as to create a transgenic
organism. Desirable transgenic plant systems having an HCV peptide
include Arabadopsis, maize, and Chlamydomonas. Desirable insect
systems having an HCV peptide include, but are not limited to, D.
melanogaster and C. elegans. Animals of any species, including, but
not limited to, amphibians, reptiles, birds, mice, hamsters, rats,
rabbits, guinea pigs, pigs, micro-pigs, goats, dogs, cats, and
non-human primates, e.g., baboons, monkeys, and chimpanzees can be
used to generate transgenic animals having an embodied HCV
molecule. These transgenic organisms desirably exhibit germline
transfer of HCV peptides described herein.
[0413] Any technique known in the art is preferably used to
introduce the HCV transgene into animals to produce the founder
lines of transgenic animals or to knock out or replace existing HCV
genes. Such techniques include, but are not limited to pronuclear
microinjection (Hoppe, P. C. and Wagner, T. E., 1989, U.S. Pat. No.
4,873,191); retrovirus mediated gene transfer into germ lines (Van
der Putten et al., Proc. Natl. Acad. Sci., USA 82:6148-6152
(1985)); gene targeting in embryonic stem cells (Thompson et al.,
Cell 56:313-321 (1989)); electroporation of embryos (Lo, Mol Cell.
Biol. 3:1803-1814 (1983); and sperm-mediated gene transfer
(Lavitrano et al., Cell 57:717-723 (1989)); see also Gordon,
Transgenic Animals, Intl. Rev. Cytol. 115:171-229 (1989).
[0414] Following synthesis or expression and isolation or
purification of the HCV peptides, the isolated or purified peptide
can be used to generate antibodies. Depending on the context, the
term "antibodies" can encompass polyclonal, monoclonal, chimeric,
single chain, Fab fragments and fragments produced by a Fab
expression library. Antibodies that recognize the HCV peptides have
many uses including, but not limited to, biotechnological
applications, therapeutic/prophylactic applications, and diagnostic
applications.
[0415] For the production of antibodies, various hosts including
goats, rabbits, rats, mice, and humans etc. can be immunized by
injection with an HCV peptide. Depending on the host species,
various adjuvants can be used to increase immunological response.
Such adjuvants include, but are not limited to, ribavirin,
Freund's, mineral gels such as aluminum hydroxide, and surface
active substances such as lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and
dinitrophenol. BCG (Bacillus Calmette-Guerin) and Corynebacterium
parvum are also potentially useful adjuvants.
[0416] Peptides used to induce specific antibodies can have an
amino acid sequence consisting of at least four amino acids, and
preferably at least 10 to 15 amino acids. By one approach, short
stretches of amino acids encoding fragments of NS3/4A are fused
with those of another protein such as keyhole limpet hemocyanin
such that an antibody is produced against the chimeric molecule.
Additionally, a composition comprising ribavirin and an HCV peptide
(SEQ. ID. NOs.: 2-11, 40-220 and SEQ. ID. NO.: 36), a fragment
thereof containing any number of consecutive amino acids between at
least 3-50 (e.g., 3, 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45,
or 50 amino acids) (e.g., SEQ. ID. NOs.: 4-26), or a nucleic acid
encoding one or more of these molecules is administered to an
animal, preferably a mammal including a human. While antibodies
capable of specifically recognizing HCV can be generated by
injecting synthetic 3-mer, 10-mer, and 15-mer peptides that
correspond to an HCV peptide into mice, a more diverse set of
antibodies can be generated by using recombinant HCV peptides,
prepared as described above.
[0417] To generate antibodies to an HCV peptide, substantially pure
peptide is isolated from a transfected or transformed cell. The
concentration of the peptide in the final preparation is adjusted,
for example, by concentration on an Amicon filter device, to the
level of a few micrograms/ml. Monoclonal or polyclonal antibody to
the peptide of interest can then be prepared as follows:
[0418] Monoclonal antibodies to an HCV peptide can be prepared
using any technique that provides for the production of antibody
molecules by continuous cell lines in culture. These include, but
are not limited to, the hybridoma technique originally described by
Koehler and Milstein (Nature 256:495-497 (1975)), the human B-cell
hybridoma technique (Kosbor et al. Immunol Today 4:72 (1983)); Cote
et al Proc Natl Acad Sci 80:2026-2030 (1983), and the EBV-hybridoma
technique Cole et al. Monoclonal Antibodies and Cancer Therapy,
Alan R. Liss Inc, New York N.Y., pp 77-96 (1985). In addition,
techniques developed for the production of "chimeric antibodies",
the splicing of mouse antibody genes to human antibody genes to
obtain a molecule with appropriate antigen specificity and
biological activity can be used. (Morrison et al. Proc Natl Acad
Sci 81:6851-6855 (1984); Neuberger et al. Nature 312:604-608(1984);
Takeda et al. Nature 314:452-454(1985)). Alternatively, techniques
described for the production of single chain antibodies (U.S. Pat.
No. 4,946,778) can be adapted to produce HCV-specific single chain
antibodies. Antibodies can also be produced by inducing in vivo
production in the lymphocyte population or by screening recombinant
immunoglobulin libraries or panels of highly specific binding
reagents as disclosed in Orlandi et al., Proc Natl Acad Sci 86:
3833-3837 (1989), and Winter G. and Milstein C; Nature 349:293-299
(1991).
[0419] Antibody fragments that contain specific binding sites for
an HCV peptide can also be generated. For example, such fragments
include, but are not limited to, the F(ab').sub.2 fragments that
can be produced by pepsin digestion of the antibody molecule and
the Fab fragments that can be generated by reducing the disulfide
bridges of the F(ab').sub.2 fragments. Alternatively, Fab
expression libraries can be constructed to allow rapid and easy
identification of monoclonal Fab fragments with the desired
specificity. (Huse W. D. et al. Science 256:1275-1281 (1989)).
[0420] By one approach, monoclonal antibodies to an HCV peptide are
made as follows. Briefly, a mouse is repetitively inoculated with a
few micrograms of the selected protein or peptides derived
therefrom over a period of a few weeks. The mouse is then
sacrificed, and the antibody producing cells of the spleen
isolated. The spleen cells are fused in the presence of
polyethylene glycol with mouse myeloma cells, and the excess
unfused cells destroyed by growth of the system on selective media
comprising aminopterin (HAT media). The successfully fused cells
are diluted and aliquots of the dilution placed in wells of a
microtiter plate where growth of the culture is continued.
Antibody-producing clones are identified by detection of antibody
in the supernatant fluid of the wells by immunoassay procedures,
such as ELISA, as originally described by Engvall, E., Meth.
Enzymol. 70:419 (1980), and derivative methods thereof. Selected
positive clones can be expanded and their monoclonal antibody
product harvested for use. Detailed procedures for monoclonal
antibody production are described in Davis, L. et al. Basic Methods
in Molecular Biology Elsevier, New York. Section 21-2.
[0421] Polyclonal antiserum containing antibodies to heterogeneous
epitopes of a single protein can be prepared by immunizing suitable
animals with the expressed protein or peptides derived therefrom
described above, which can be unmodified or modified to enhance
immunogenicity. Effective polyclonal antibody production is
affected by many factors related both to the antigen and the host
species. For example, small molecules tend to be less immunogenic
than others and can require the use of carriers and adjuvant. Also,
host animals vary in response to site of inoculations and dose,
with both inadequate or excessive doses of antigen resulting in low
titer antisera. Small doses (ng level) of antigen administered at
multiple intradermal sites appears to be most reliable. An
effective immunization protocol for rabbits can be found in
Vaitukaitis, J. et al. J Clin. Endocrinol. Metab. 33:988-991
(1971).
[0422] Booster injections are given at regular intervals, and
antiserum harvested when antibody titer thereof, as determined
semi-quantitatively, for example, by double immunodiffusion in agar
against known concentrations of the antigen, begins to fall. See,
for example, Ouchterlony, O. et al., Chap. 19 in: Handbook of
Experimental Immunology D. Wier (ed) Blackwell (1973). Plateau
concentration of antibody is usually in the range of 0.1 to 0.2
mg/ml of serum (about 12 .mu.M). Affinity of the antisera for the
antigen is determined by preparing competitive binding curves, as
described, for example, by Fisher, D., Chap. 42 in: Manual of
Clinical Immunology, 2d Ed. (Rose and Friedman, Eds.) Amer. Soc.
For Microbiol., Washington, D.C. (1980). Antibody preparations
prepared according to either protocol are useful in quantitative
immunoassays that determine concentrations of antigen-bearing
substances in biological samples; they are also used
semi-quantitatively or qualitatively (e.g., in diagnostic
embodiments that identify the presence of HCV in biological
samples). The next section describes how some of the novel nucleic
acids and peptides described above can be used in diagnostics.
Diagnostic Embodiments
[0423] Generally, the embodied diagnostics are classified according
to whether a nucleic acid or protein-based assay is used. Some
diagnostic assays detect the presence or absence of an embodied HCV
nucleic acid sequence in a sample obtained from a patient, whereas,
other assays seek to identify whether an embodied HCV peptide is
present in a biological sample obtained from a patient.
Additionally, the manufacture of kits that incorporate the reagents
and methods described herein that allow for the rapid detection and
identification of HCV are also embodied. These diagnostic kits can
include, for example, an embodied nucleic acid probe or antibody,
which specifically detects HCV. The detection component of these
kits will typically be supplied in combination with one or more of
the following reagents. A support capable of absorbing or otherwise
binding DNA, RNA, or protein will often be supplied. Available
supports include membranes of nitrocellulose, nylon or derivatized
nylon that can be characterized by bearing an array of positively
charged substituents. One or more restriction enzymes, control
reagents, buffers, amplification enzymes, and non-human
polynucleotides like calf-thymus or salmon-sperm DNA can be
supplied in these kits.
[0424] Useful nucleic acid-based diagnostics include, but are not
limited to, direct DNA sequencing, Southern Blot analysis, dot blot
analysis, nucleic acid amplification, and combinations of these
approaches. The starting point for these analysis is isolated or
purified nucleic acid from a biological sample obtained from a
patient suspected of contracting HCV or a patient at risk of
contracting HCV. The nucleic acid is extracted from the sample and
can be amplified by RT-PCR and/or DNA amplification using primers
that correspond to regions flanking the embodied HCV nucleic acid
sequences (e.g., NS3/4A (SEQ. ID. NO.: 1)).
[0425] In some embodiments, nucleic acid probes that specifically
hybridize with HCV sequences are attached to a support in an
ordered array, wherein the nucleic acid probes are attached to
distinct regions of the support that do not overlap with each
other. Preferably, such an ordered array is designed to be
"addressable" where the distinct locations of the probe are
recorded and can be accessed as part of an assay procedure. These
probes are joined to a support in different known locations. The
knowledge of the precise location of each nucleic acid probe makes
these "addressable" arrays particularly useful in binding assays.
The nucleic acids from a preparation of several biological samples
are then labeled by conventional approaches (e.g., radioactivity or
fluorescence) and the labeled samples are applied to the array
under conditions that permit hybridization.
[0426] If a nucleic acid in the samples hybridizes to a probe on
the array, then a signal will be detected at a position on the
support that corresponds to the location of the hybrid. Since the
identity of each labeled sample is known and the region of the
support on which the labeled sample was applied is known, an
identification of the presence of the polymorphic variant can be
rapidly determined. These approaches are easily automated using
technology known to those of skill in the art of high throughput
diagnostic or detection analysis.
[0427] Additionally, an approach opposite to that presented above
can be employed. Nucleic acids present in biological samples can be
disposed on a support so as to create an addressable array.
Preferably, the samples are disposed on the support at known
positions that do not overlap. The presence of HCV nucleic acids in
each sample is determined by applying labeled nucleic acid probes
that complement nucleic acids, which encode HCV peptides, at
locations on the array that correspond to the positions at which
the biological samples were disposed. Because the identity of the
biological sample and its position on the array is known, the
identification of a patient that has been infected with HCV can be
rapidly determined. These approaches are also easily automated
using technology known to those of skill in the art of high
throughput diagnostic analysis.
[0428] Any addressable array technology known in the art can be
employed. One particular embodiment of polynucleotide arrays is
known as Genechips.TM., and has been generally described in U.S.
Pat. No. 5,143,854; PCT publications WO 90/15070 and 92/10092.
These arrays are generally produced using mechanical synthesis
methods or light directed synthesis methods, which incorporate a
combination of photolithographic methods and solid phase
oligonucleotide synthesis. (Fodor et al., Science, 251:767-777,
(1991)). The immobilization of arrays of oligonucleotides on solid
supports has been rendered possible by the development of a
technology generally identified as "Very Large Scale Immobilized
Polymer Synthesis" (VLSPIS.TM.) in which, typically, probes are
immobilized in a high density array on a solid surface of a chip.
Examples of VLSPIS.TM. technologies are provided in U.S. Pat. Nos.
5,143,854 and 5,412,087 and in PCT Publications WO 90/15070, WO
92/10092 and WO 95/11995, which describe methods for forming
oligonucleotide arrays through techniques such as light-directed
synthesis techniques. In designing strategies aimed at providing
arrays of nucleotides immobilized on solid supports, further
presentation strategies were developed to order and display the
oligonucleotide arrays on the chips in an attempt to maximize
hybridization patterns and diagnostic information. Examples of such
presentation strategies are disclosed in PCT Publications WO
94/12305, WO 94/11530, WO 97/29212, and WO 97/31256.
[0429] A wide variety of labels and conjugation techniques are
known by those skilled in the art and can be used in various
nucleic acid assays. There are several ways to produce labeled
nucleic acids for hybridization or PCR including, but not limited
to, oligolabeling, nick translation, end-labeling, or PCR
amplification using a labeled nucleotide. Alternatively, a nucleic
acid encoding an HCV peptide can be cloned into a vector for the
production of an mRNA probe. Such vectors are known in the art, are
commercially available, and can be used to synthesize RNA probes in
vitro by addition of an appropriate RNA polymerase such as T7, T3
or SP6 and labeled nucleotides. A number of companies such as
Pharmacia Biotech (Piscataway N.J.), Promega (Madison Wis.), and
U.S. Biochemical Corp (Cleveland Ohio) supply commercial kits and
protocols for these procedures. Suitable reporter molecules or
labels include those radionuclides, enzymes, fluorescent,
chemiluminescent, or chromogenic agents, as well as, substrates,
cofactors, inhibitors, magnetic particles and the like.
[0430] The presence of an HCV peptide in a protein sample obtained
from a patient can also be detected by using conventional assays
and the embodiments described herein. For example, antibodies that
are immunoreactive with the disclosed HCV peptides can be used to
screen biological samples for the presence of HCV infection. In
preferred embodiments, antibodies that are reactive to the embodied
HCV peptides are used to immunoprecipitate the disclosed HCV
peptides from biological samples or are used to react with proteins
obtained from a biological sample on Western or Immunoblots.
Favored diagnostic embodiments also include enzyme-linked
immunosorbant assays (ELISA), radioimmunoassays (RIA),
immunoradiometric assays (IRMA) and immunoenzymatic assays (IEMA),
including sandwich assays using monoclonal and/or polyclonal
antibodies specific for the disclosed HCV peptides. Exemplary
sandwich assays are described by David et al., in U.S. Pat. Nos.
4,376,110 and 4,486,530. Other embodiments employ aspects of the
immune-strip technology disclosed in U.S. Pat. Nos. 5,290,678;
5,604,105; 5,710,008; 5,744,358; and 5,747,274.
[0431] In another preferred protein-based diagnostic, the
antibodies described herein are attached to a support in an ordered
array, wherein a plurality of antibodies are attached to distinct
regions of the support that do not overlap with each other. As with
the nucleic acid-based arrays, the protein-based arrays are ordered
arrays that are designed to be "addressable" such that the distinct
locations are recorded and can be accessed as part of an assay
procedure. These probes are joined to a support in different known
locations. The knowledge of the precise location of each probe
makes these "addressable" arrays particularly useful in binding
assays. For example, an addressable array can comprise a support
having several regions to which are joined a plurality of antibody
probes that specifically recognize HCV peptides present in a
biological sample and differentiate the isotype of HCV identified
herein.
[0432] By one approach, proteins are obtained from biological
samples and are then labeled by conventional approaches (e.g.,
radioactivity, colorimetrically, or fluorescently). The labeled
samples are then applied to the array under conditions that permit
binding. If a protein in the sample binds to an antibody probe on
the array, then a signal will be detected at a position on the
support that corresponds to the location of the antibody-protein
complex. Since the identity of each labeled sample is known and the
region of the support on which the labeled sample was applied is
known, an identification of the presence, concentration, and/or
expression level can be rapidly determined. That is, by employing
labeled standards of a known concentration of HCV peptide, an
investigator can accurately determine the protein concentration of
the particular peptide in a tested sample and can also assess the
expression level of the HCV peptide. Conventional methods in
densitometry can also be used to more accurately determine the
concentration or expression level of the HCV peptide. These
approaches are easily automated using technology known to those of
skill in the art of high throughput diagnostic analysis.
[0433] In another embodiment, an approach opposite to that
presented above can be employed. Proteins present in biological
samples can be disposed on a support so as to create an addressable
array. Preferably, the protein samples are disposed on the support
at known positions that do not overlap. The presence of an HCV
peptide in each sample is then determined by applying labeled
antibody probes that recognize epitopes specific for the HCV
peptide. Because the identity of the biological sample and its
position on the array is known, an identification of the presence,
concentration, and/or expression level of an HCV peptide can be
rapidly determined.
[0434] That is, by employing labeled standards of a known
concentration of HCV peptide, an investigator can accurately
determine the concentration of peptide in a sample and from this
information can assess the expression level of the peptide.
Conventional methods in densitometry can also be used to more
accurately determine the concentration or expression level of the
HCV peptide. These approaches are also easily automated using
technology known to those of skill in the art of high throughput
diagnostic analysis. As detailed above, any addressable array
technology known in the art can be employed. The next section
describes more compositions that include the HCV nucleic acids
and/or HCV peptides described herein.
Compositions Comprising HCV Nucleic Acids or Peptides
[0435] Embodiments of the invention also include NS3/4A fusion
proteins or nucleic acids encoding these molecules. For instance,
production and purification of recombinant protein may be
facilitated by the addition of auxiliary amino acids to form a
"tag". Such tags include, but are not limited to, His-6, Flag, Myc
and GST. The tags may be added to the C-terminus, N-terminus, or
within the NS3/4A amino acid sequence. Further embodiments include
NS3/4A fusion proteins with amino or carboxy terminal truncations,
or internal deletions, or with additional polypeptide sequences
added to the amino or carboxy terminal ends, or added internally.
Other embodiments include NS3/4A fusion proteins, or truncated or
mutated versions thereof, where the residues of the NS3/4A
proteolytic cleavage site have been substituted. Such substitutions
include, but are not limited to, sequences where the P1' site is a
Ser, Gly, or Pro, or the P1 position is an Arg, or where the P8 to
P4' sequence is Ser-Ala-Asp-Leu-Glu-Val-Val-Thr-Ser-Thr-Trp-Val
(SEQ. ID. NO.: 15).
[0436] More embodiments concern an immunogen comprising the NS3/4A
fusion protein, or a truncated, mutated, or modified version
thereof, capable of eliciting an enhanced immune response against
NS3. The immunogen can be provided in a substantially purified
form, which means that the immunogen has been rendered
substantially free of other proteins, lipids, carbohydrates or
other compounds with which it naturally associates.
[0437] Some embodiments contain at least one of the HCV nucleic
acids or HCV peptides (e.g., SEQ. ID. NOs.: 1-27, 35, 36 or 40-220)
joined to a support. Preferably, these supports are manufactured so
as to create a multimeric agent. These multimeric agents provide
the HCV peptide or nucleic acid in such a form or in such a way
that a sufficient affinity to the molecule is achieved. A
multimeric agent having an HCV nucleic acid or peptide can be
obtained by joining the desired molecule to a macromolecular
support. A "support" can be a termed a carrier, a protein, a resin,
a cell membrane, a capsid or portion thereof, or any macromolecular
structure used to join or immobilize such molecules. Solid supports
include, but are not limited to, the walls of wells of a reaction
tray, test tubes, polystyrene beads, magnetic beads, nitrocellulose
strips, membranes, microparticles such as latex particles, animal
cells, Duracyte.RTM., artificial cells, and others. An HCV nucleic
acid or peptide can also be joined to inorganic carriers, such as
silicon oxide material (e.g., silica gel, zeolite, diatomaceous
earth or aminated glass) by, for example, a covalent linkage
through a hydroxy, carboxy or amino group and a reactive group on
the carrier.
[0438] In several multimeric agents, the macromolecular support has
a hydrophobic surface that interacts with a portion of the HCV
nucleic acid or peptide by a hydrophobic non-covalent interaction.
In some cases, the hydrophobic surface of the support is a polymer
such as plastic or any other polymer in which hydrophobic groups
have been linked such as polystyrene, polyethylene or polyvinyl.
Additionally, HCV nucleic acid or peptide can be covalently bound
to carriers including proteins and oligo/polysaccarides (e.g.
cellulose, starch, glycogen, chitosane or aminated sepharose). In
these later multimeric agents, a reactive group on the molecule,
such as a hydroxy or an amino group, is used to join to a reactive
group on the carrier so as to create the covalent bond. Additional
multimeric agents comprise a support that has other reactive groups
that are chemically activated so as to attach the HCV nucleic acid
or peptide. For example, cyanogen bromide activated matrices, epoxy
activated matrices, thio and thiopropyl gels, nitrophenyl
chloroformate and N-hydroxy succinimide chlorformate linkages, or
oxirane acrylic supports are used. (Sigma).
[0439] Carriers for use in the body, (i.e. for prophylactic or
therapeutic applications) are desirably physiological, non-toxic
and preferably, non-immunoresponsive. Suitable carriers for use in
the body include poly-L-lysine, poly-D, L-alanine, liposomes,
capsids that display the desired HCV peptide or nucleic acid, and
Chromosorb.RTM. (Johns-Manville Products, Denver Co.). Ligand
conjugated Chromosorb.RTM. (Synsorb-Pk) has been tested in humans
for the prevention of hemolytic-uremic syndrome and was reported as
not presenting adverse reactions. (Armstrong et al. J. Infectious
Diseases 171:1042-1045 (1995)). For some embodiments, a "naked"
carrier (i.e., lacking an attached HCV nucleic acid or peptide)
that has the capacity to attach an HCV nucleic acid or peptide in
the body of a organism is administered. By this approach, a
"prodrug-type" therapy is envisioned in which the naked carrier is
administered separately from the HCV nucleic acid or peptide and,
once both are in the body of the organism, the carrier and the HCV
nucleic acid or peptide are assembled into a multimeric
complex.
[0440] The insertion of linkers, (e.g., ".lamda. linkers"
engineered to resemble the flexible regions of .lamda. phage) of an
appropriate length between the HCV nucleic acid or peptide and the
support are also contemplated so as to encourage greater
flexibility of the HCV peptide, hybrid, or binding partner and
thereby overcome any steric hindrance that can be presented by the
support. The determination of an appropriate length of linker that
allows for an optimal cellular response or lack thereof, can be
determined by screening the HCV nucleic acid or peptide with
varying linkers in the assays detailed in the present
disclosure.
[0441] A composite support comprising more than one type of HCV
nucleic acid or peptide is also envisioned. A "composite support"
can be a carrier, a resin, or any macromolecular structure used to
attach or immobilize two or more different HCV nucleic acids or
peptides. As above, the insertion of linkers, such as .lamda.
linkers, of an appropriate length between the HCV nucleic acid or
peptide and the support is also contemplated so as to encourage
greater flexibility in the molecule and thereby overcome any steric
hindrance that can occur. The determination of an appropriate
length of linker that allows for an optimal cellular response or
lack thereof, can be determined by screening the HCV nucleic acid
or peptide with varying linkers in the assays detailed in the
present disclosure.
[0442] In other embodiments, the multimeric and composite supports
discussed above can have attached multimerized HCV nucleic acids or
peptides so as to create a "multimerized-multimeric support" and a
"multimerized-composite support", respectively. A multimerized
ligand can, for example, be obtained by coupling two or more HCV
nucleic acids or peptides in tandem using conventional techniques
in molecular biology. The multimerized form of the HCV nucleic acid
or peptide can be advantageous for many applications because of the
ability to obtain an agent with a higher affinity, for example. The
incorporation of linkers or spacers, such as flexible .lamda.
linkers, between the individual domains that make-up the
multimerized agent can also be advantageous for some embodiments.
The insertion of .lamda. linkers of an appropriate length between
protein binding domains, for example, can encourage greater
flexibility in the molecule and can overcome steric hindrance.
Similarly, the insertion of linkers between the multimerized HCV
nucleic acid or peptide and the support can encourage greater
flexibility and limit steric hindrance presented by the support.
The determination of an appropriate length of linker can be
determined by screening the HCV nucleic acids or peptides in the
assays detailed in this disclosure.
[0443] Embodiments also include vaccine compositions and immunogen
preparations comprising the NS3/4A fusion protein, or a truncated
or mutated version thereof, and, optionally, an adjuvant. The next
section describes some of these compositions in greater detail.
[0444] Vaccine Compositions and Immunogenic Preparations
[0445] Vaccine compositions and immunogenic preparations
comprising, consisting of, or consisting essentially of either an
embodied nucleic acid encoding a chimeric NS3/4A peptide or a
chimeric NS3/4A polypeptide, or both, are contemplated. These
compositions typically contain an adjuvant, but do not necessarily
require an adjuvant. That is many of the nucleic acids and peptides
described herein function as immunogens when administered neat. The
compositions described herein (e.g., the NS3/4A chimeric immunogens
and vaccine compositions containing an adjuvant, such as ribavirin)
can be manufactured in accordance with conventional methods of
galenic pharmacy to produce medicinal agents for administration to
animals, e.g., mammals including humans. (See, e.g., U.S. Pat. Nos.
6,680,059 and 6,858,590, hereby expressly incorporated by reference
in their entireties).
[0446] Various nucleic acid-based vaccines are known and it is
contemplated that these compositions and approaches to
immunotherapy can be augmented by reformulation with ribavirin
(See, e.g., U.S. Pat. Nos. 5,589,466 and 6,235,888, hereby
expressly incorporated by reference in their entireties). By one
approach, for example, a gene encoding one of the NS3/4A chimeric
polypeptides described herein is cloned into an expression vector
capable of expressing the polypeptide when introduced into a
subject. The expression construct is introduced into the subject in
a mixture of adjuvant (e.g., ribavirin) or in conjunction with an
adjuvant (e.g., ribavirin). For example, the adjuvant (e.g.,
ribavirin) is administered shortly after the expression construct
at the same site. Alternatively, RNA encoding the NS3/4A chimeric
polypeptide of interest is provided to the subject in a mixture
with ribavirin or in conjunction with an adjuvant (e.g.,
ribavirin).
[0447] Where the antigen is to be DNA (e.g., preparation of a DNA
vaccine composition), suitable promoters include Simian Virus 40
(SV40), Mouse Mammary Tumor Virus (MMTV) promoter, Human
Immunodeficiency Virus (HIV) such as the HIV Long Terminal Repeat
(LTR) promoter, Moloney virus, ALV, Cytomegalovirus (CMV) such as
the CMV immediate early promoter, Epstein Barr Virus (EBV), Rous
Sarcoma Virus (RSV) as well as promoters from human genes such as
human actin, human myosin, human hemoglobin, human muscle creatine
and human metalothionein can be used. Examples of polyadenylation
signals useful with some embodiments, especially in the production
of a genetic vaccine for humans, include but are not limited to,
SV40 polyadenylation signals and LTR polyadenylation signals. In
particular, the SV40 polyadenylation signal, which is in pCEP4
plasmid (Invitrogen, San Diego Calif.), referred to as the SV40
polyadenylation signal, is used.
[0448] In addition to the regulatory elements required for gene
expression, other elements may also be included in a gene
construct. Such additional elements include enhancers. The enhancer
may be selected from the group including but not limited to: human
actin, human myosin, human hemoglobin, human muscle creatine and
viral enhancers such as those from CMV, RSV and EBV. Gene
constructs can be provided with mammalian origin of replication in
order to maintain the construct extrachromosomally and produce
multiple copies of the construct in the cell. Plasmids pCEP4 and
pREP4 from Invitrogen (San Diego, Calif.) contain the Epstein Barr
virus origin of replication and nuclear antigen EBNA-1 coding
region, which produces high copy episomal replication without
integration. All forms of DNA, whether replicating or
non-replicating, which do not become integrated into the genome,
and which are expressible, can be used. Preferably, the genetic
vaccines comprise ribavirin and a nucleic acid encoding a NS3/4A
polypeptide.
[0449] More embodiments concern an immunogen comprising the
chimeric NS3/4A polypeptide, or a truncated, mutated, or modified
version thereof, capable of eliciting an enhanced immune response
against a target antigen. The immunogen can be provided in a
substantially purified form, which means that the immunogen has
been rendered substantially free of other proteins, lipids,
carbohydrates or other compounds with which it naturally
associates.
[0450] Some embodiments contain at least one of the nucleic acids
described joined to a support. Preferably, these supports are
manufactured so as to create a multimeric agent. These multimeric
agents provide the chimeric NS3/4A chimeric polypeptide or encoding
nucleic acid in such a form or in such a way that a sufficient
affinity to the molecule is achieved. A multimeric agent having a
chimeric NS3/4A chimeric polypeptide or encoding nucleic acid can
be obtained by joining the desired molecule to a macromolecular
support. A "support" can be a termed a carrier, a protein, a resin,
a cell membrane, a capsid or portion thereof, or any macromolecular
structure used to join or immobilize such molecules. Solid supports
include, but are not limited to, the walls of wells of a reaction
tray, test tubes, polystyrene beads, magnetic beads, nitrocellulose
strips, membranes, microparticles such as latex particles, animal
cells, DURACYTE.RTM., artificial cells, and others. A chimeric
NS3/4A polypeptide or encoding nucleic acid can also be joined to
inorganic carriers, such as silicon oxide material (e.g., silica
gel, zeolite, diatomaceous earth or aminated glass) by, for
example, a covalent linkage through a hydroxy, carboxy or amino
group and a reactive group on the carrier.
[0451] In several multimeric agents, the macromolecular support has
a hydrophobic surface that interacts with a portion of the chimeric
NS3/4A chimeric polypeptide or encoding nucleic acid by a
hydrophobic non-covalent interaction. In some cases, the
hydrophobic surface of the support is a polymer such as plastic or
any other polymer in which hydrophobic groups have been linked such
as polystyrene, polyethylene or polyvinyl. Additionally, chimeric
NS3/4A polypeptides or encoding nucleic acids can be covalently
bound to carriers including proteins and oligo/polysaccharides
(e.g. cellulose, starch, glycogen, chitosane, aminated sepharose,
or the gal epitope (e.g., gal-.alpha.-1, 3 gal-.beta.). In these
later multimeric agents, a reactive group on the molecule, such as
a hydroxy or an amino group, is used to join to a reactive group on
the carrier so as to create the covalent bond. Additional
multimeric agents comprise a support that has other reactive groups
that are chemically activated so as to attach chimeric NS3/4A
polypeptides or encoding nucleic acids. For example, cyanogen
bromide activated matrices, epoxy activated matrices, thio and
thiopropyl gels, nitrophenyl chloroformate and N-hydroxy
succinimide chlorformate linkages, or oxirane acrylic supports are
used. (Sigma).
[0452] Carriers for use in the body, (i.e. for prophylactic or
therapeutic applications) are desirably physiological, non-toxic
and preferably, non-immunoresponsive. Suitable carriers for use in
the body include poly-L-lysine, poly-D, L-alanine, liposomes,
capsids that display the desired NS3/4A chimeric peptide or nucleic
acid, and CHROMSORB.RTM. (Johns-Manville Products, Denver Co.).
Ligand conjugated CHROMSORB.RTM. (Synsorb-Pk) has been tested in
humans for the prevention of hemolytic-uremic syndrome and was
reported as not presenting adverse reactions. (Armstrong et al. J.
Infectious Diseases 171:1042-1045 (1995)). For some embodiments, a
"naked" carrier (i.e., lacking an attached chimeric NS3/4A chimeric
polypeptides or encoding nucleic acids) that has the capacity to
attach a chimeric NS3/4A chimeric polypeptide or encoding nucleic
acid in the body of a organism is administered. By this approach, a
"prodrug-type" therapy is envisioned in which the naked carrier is
administered separately from the NS3/4A chimeric polypeptide or
encoding nucleic acid and, once both are in the body of the
organism, the carrier and NS3/4A chimeric polypeptide or encoding
nucleic acid are assembled into a multimeric complex.
[0453] The insertion of linkers of an appropriate length between
the NS3/4A chimeric polypeptide or encoding nucleic acid and the
support are also contemplated so as to encourage greater
flexibility of the NS3/4A chimeric polypeptide, encoding nucleic
acid, hybrid, or binding partner and thereby overcome any steric
hindrance that can be presented by the support. The determination
of an appropriate length of linker that allows for an optimal
cellular response or lack thereof, can be determined by screening
the NS3/4A chimeric polypeptide or encoding nucleic acid with
varying linkers in the assays detailed in the present
disclosure.
[0454] A composite support comprising more than one type of NS3/4A
chimeric polypeptide or encoding nucleic acid is also envisioned. A
"composite support" can be a carrier, a resin, or any
macromolecular structure used to attach or immobilize two or more
different NS3/4A chimeric polypeptides or encoding nucleic acids.
As above, the insertion of linkers, such as .lamda. linkers, of an
appropriate length between the NS3/4A chimeric polypeptide or
encoding nucleic acid and the support is also contemplated so as to
encourage greater flexibility in the molecule and thereby overcome
any steric hindrance that can occur. The determination of an
appropriate length of linker that allows for an optimal cellular
response or lack thereof, can be determined by screening the NS3/4A
chimeric polypeptide or encoding nucleic acid with varying linkers
in the assays detailed in the present disclosure.
[0455] In other embodiments, the multimeric and composite supports
discussed above can have attached multimerized NS3/4A chimeric
polypeptides or encoding nucleic acids so as to create a
"multimerized-multimeric support" and a "multimerized-composite
support", respectively. A multimerized ligand can, for example, be
obtained by coupling two or more NS3/4A chimeric polypeptides or
encoding nucleic acids in tandem using conventional techniques in
molecular biology. The multimerized form of NS3/4A chimeric
polypeptides or encoding nucleic acids can be advantageous for many
applications because of the ability to obtain an agent with a
higher affinity, for example. The incorporation of linkers such as
flexible .lamda. linkers, between the individual domains that
make-up the multimerized agent can also be advantageous for some
embodiments. The insertion of .lamda. linkers of an appropriate
length between protein binding domains, for example, can encourage
greater flexibility in the molecule and can overcome steric
hindrance. Similarly, the insertion of linkers between the
multimerized NS3/4A chimeric polypeptides or encoding nucleic acids
and the support can encourage greater flexibility and limit steric
hindrance presented by the support. The determination of an
appropriate length of linker can be determined by screening the
NS3/4A chimeric polypeptides or encoding nucleic acid in the assays
detailed in this disclosure.
[0456] Aspects of the present invention also relate to a modified
hepatitis C virus (HCV) NS3/4A protein that is linked to a pendent
hapten (e.g., a T cell epitope) through chemically-reactive amino
acid residue, which may be, optionally, inserted into or attached
to (e.g., a linker on either the N terminal or C terminal end) the
NS3/4A sequence. Such an introduced or already present
chemically-reactive amino acid residue is characterized in that it
has a chemically-reactive side chain that provides a chemical group
for pendently linking the NS3/4A polypeptide to the hapten.
Typically, the chemically-reactive amino acid residue is a lysine,
cysteine, or histidine residue or a carboxyl-containing residue
such as aspartic acid or glutamic acid, preferably lysine or a
carboxyl-containing residue, and most preferably lysine. The hapten
bonded to the chemically-reactive amino acid residue is any
compound of interest for generating an immune response, and is
typically a T cell epitope. Preferably, the hapten is a polypeptide
hapten, a carbohydrate hapten, or a non-peptidal/non-saccharidal
(chemical) hapten. In some embodiments, the hapten is a
pathogen-related hapten, for example, a T cell epitope provided
herein. The word "hapten" is used in this context to describe
molecules that are capable of stimulating an immune response (e.g.,
production of antibody) when chemically coupled to NS3/4A. The word
is often used for small nonantigenic molecules in the art, but
herein, it merely refers to the molecule that is to be pendently
linked to NS3/4A, even if it is antigenic or not small. The
chemically-reactive amino acid residue can be at any position
within the epitope and NS3/4A polypeptide or attached to either end
of these molecules (N terminal or C terminal). As stated above, the
introduced chemically-reactive amino acid residue has a
chemically-reactive side chain that provides a functional group for
derivatizing the NS3/4A polypeptide (e.g., conjugating a T cell
epitope to the NS3/4A). Useful side chain functional groups include
epsilon-amino groups, beta- or gamma-carboxyl groups, thiol (--SH)
groups and aromatic rings (e.g. tyrosine and histidine). The
chemically-reactive amino acid residue is typically a lysine,
cysteine, or histidine residue or a carboxyl-containing residue
such as aspartic acid or glutamic acid. Lysine is a particularly
preferred chemically-reactive amino acid residue. In addition of
the use of an individual chemically-reactive amino acid residue in
the insert such as aspartic acid or lysine, substantially any
sequence of the desired length that contains a chemically-reactive
amino acid residue can be used.
[0457] Any hapten against which antibody production is desired can
be linked to an NS3/4A peptide or fragment thereof, as described
herein, to form an immunogenic composition. The hapten of interest
typically is T cell epitope. The hapten can be a polypeptide, a
carbohydrate (saccharide), or a non-polypeptide, non-carbohydrate
chemical such as 2,4-dinitrobenzene, however.
[0458] Methods for operatively linking individual haptens to a
protein or polypeptide through an amino acid residue side chain of
the protein or polypeptide to form a pendently-linked immunogenic
conjugate, e.g., a branched-chain polypeptide polymer, are well
known in the art. Those methods include linking through one or more
types of functional groups on various side chains and result in the
carrier protein polypeptide backbone being pendently
linked--covalently linked (coupled) to the hapten but separated by
at least one side chain.
[0459] Methods for linking carrier proteins to haptens using each
of the above functional groups are described in Erlanger, Method of
Enzymology, 70:85 (1980), Aurameas, et al., Scand. J. Immunol.,
Vol. 8, Suppl. 7, 7-23 (1978) and U.S. Pat. No. 4,493,795 to Nestor
et al., all of which are hereby expressly incorporated by reference
in their entireties. In addition, a site-directed coupling
reaction, as described in Rodwell et al., Biotech., 3, 889-894
(1985), herein expressly incorporated by reference in its entirety,
can be carried out so that the biological activity of the
polypeptides is not substantially diminished.
[0460] Furthermore, as is well known in the art, both the NS3/4A
protein or fragment thereof and a polypeptide hapten can be used in
their native form or their functional group content can be modified
by succinylation of lysine residues or reaction with
cysteine-thiolactone. A sulfhydryl group can also be incorporated
into either molecule by reaction of amino functions with
2-iminothiolane or the N-hydroxysuccinimide ester of
3-(3-dithiopyridyl)propionate.
[0461] The NS3/4A peptide or fragment thereof or hapten can also be
modified to incorporate a spacer arm, such as hexamethylene diamine
or other bifunctional molecules of similar size, to facilitate the
pendent linking.
[0462] Methods for covalent bonding of a polypeptide hapten are
extremely varied and are well known by workers skilled in the
immunological arts. For example, following U.S. Pat. No. 4,818,527,
m-maleimidobenzoyl-N-hydroxysuccinimde ester (ICN Biochemicals,
Inc.) or succinimidyl
4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC, Pierce), all
of which are hereby expressly incorporated by reference in their
entireties, is reacted with a NS3/4A protein or fragment thereof to
form an activated molecule. That activated carrier is then reacted
with a polypeptide that either contains a terminal cysteine or to
which an additional amino- or carboxy-terminal cysteine residue has
been added to form a covalently bonded NS3/4A conjugate. As an
alternative example, the amino group of a polypeptide hapten can be
first reacted with N-succinimidyl 3-(2-pyridylthio)propionate
(SPDP, Pharmacia), and that thiol-containing polypeptide can be
reacted with the activated NS3/4A after reduction. Of course, the
sulfur-containing moiety and double bond-containing Michael
acceptor can be reversed. These reactions are described in the
supplier's literature, and also in Kitagawa, et al., J. Biochem.,
79:233 (1976) and in Lachmann et al., in 1986 Synthetic Peptides as
Antigens, (Ciba Foundation Symposium 119), pp. 25-40 (Wiley,
Chichester: 1986), all of which are hereby expressly incorporated
by reference in their entireties.
[0463] U.S. Pat. No. 4,767,842, herein expressly incorporated by
reference in its entirety, teaches several modes of covalent
attachment between a carrier and polypeptide that are useful here.
In one method, tolylene diisocyanate is reacted with the NS3/4A or
a fragment thereof in a dioxane-buffer solvent at zero degrees C.
to form an activated molecule. A polypeptide hapten (e.g., a T cell
epitope) is thereafter admixed and reacted with the activated
NS3/4A to form the covalently bonded NS3/4A conjugate.
[0464] Particularly useful are a large number of heterobifunctional
agents that form a disulfide link at one functional group end and a
peptide link at the other, including
N-succidimidyl-3-(2-pyridyldithio) propionate (SPDP). This reagent
creates a disulfide linkage between itself and a thiol in either
the NS3/4A or fragment thereof or the hapten, for example a
cysteine residue in a polypeptide hapten, and an amide linkage on
the coupling partner, for example the amino on a lysine or other
free amino group in the NS3/4A. A variety of such disulfide/amide
forming agents are known. (See for example Immun. Rev. (1982)
62:185, herein expressly incorporated by reference in its
entirety). Other bifunctional coupling agents form a thioether
rather than a disulfide linkage. Many of these thioether-forming
agents are commercially available and include reactive esters of
6-maleimidocaproic acid, 2-bromoacetic acid, 2-iodoacetic acid,
4-(N-maleimido-methyl)cyclohexane-1-carboxylic acid and the like.
The carboxyl groups can be activated by combining them with
succinimide or 1-hydroxy-2-nitro-4-sulfonic acid, sodium salt. The
particularly preferred coupling agent is succinimidyl
4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) obtained
from Pierce Company, Rockford, Ill. The foregoing list is not meant
to be exhaustive, and modifications of the named compounds can
clearly be used.
[0465] A polypeptide hapten (e.g., a T cell epitope) can be
obtained in a number of ways well known in the art. Usual peptide
synthesis techniques can be readily utilized. For example,
recombinant and PCR-based techniques to produce longer peptides are
useful. Because the desired sequences are usually relatively short,
solid phase chemical synthesis is useful.
[0466] As discussed below, DNA sequences that encode a variety of
polypeptide haptens (e.g., T cell epitopes) are known in the art.
The coding sequence for peptides of the length contemplated herein
can easily be synthesized by chemical techniques, for example, the
phosphotriester method of Matteucci et al., J. Am. Chem. Soc.
103:3185 (1981). Of course, by chemically synthesizing the coding
sequence, any desired modification can be made simply by
substituting the appropriate bases for those encoding the native
peptide sequence. The coding sequence can then be provided with
appropriate linkers and ligated into expression vectors now
commonly available in the art, and the regulating vectors used to
transform suitable hosts to produce the desired protein.
[0467] A number of such vectors and suitable host systems are now
available. For example promoter sequences compatible with bacterial
hosts are provided in plasmids containing convenient restriction
sites for insertion of the desired coding sequence. Typical of such
vector plasmids are, for example, pUC8, and pUC13 available from J.
Messing, at the University of Minnesota (see, e.g., Messing et al.,
Nucleic Acids Res. 9:309 (1981)) or pBR322, available from New
England Biolabs. Suitable promoters include, for example, the
beta-lactamase (penicillinase) and lactose (lac) promoter systems
(Chang. et al., Nature 198:1056 (1977) and the tryptophan (trp)
promoter system (Goeddel et al., Nucleic Acids Res. 8:4057 (1980)).
The resulting expression vectors are transformed into suitable
bacterial hosts using the calcium chloride method described by
Cohen, et al., Proc. Natl. Acad. Sci. U.S.A. 69:2110 (1972).
Successful transformants may produce the desired polypeptide
fragments at higher levels than those found in strains normally
producing the intact pili. Of course, yeast or mammalian cell hosts
can also be used, employing suitable vectors and control
sequences.
[0468] Embodiments also include methods of using vaccine
compositions and immunogen preparations comprising the NS3/4A
chimeric polypeptides or encoding nucleic acids, or a truncated or
mutated version thereof, and, optionally, an adjuvant. The next
section describes some of these compositions in greater detail.
[0469] Methods of Using the Vaccine Compositions and Immunogen
Preparations
[0470] Routes of administration of the embodiments described herein
include, but are not limited to, transdermal, parenteral,
gastrointestinal, transbronchial, and transalveolar. Transdermal
administration can be accomplished by application of a cream,
rinse, gel, etc. capable of allowing the compositions described
herein to penetrate the skin. Parenteral routes of administration
include, but are not limited to, electrical or direct injection
such as direct injection into a central venous line, intravenous,
intramuscular, intraperitoneal, intradermal, or subcutaneous
injection. Gastrointestinal routes of administration include, but
are not limited to, ingestion and rectal. Transbronchial and
transalveolar routes of administration include, but are not limited
to, inhalation, either via the mouth or intranasally.
[0471] Compositions that are suitable for transdermal
administration include, but are not limited to, pharmaceutically
acceptable suspensions, oils, creams, and ointments applied
directly to the skin or incorporated into a protective carrier such
as a transdermal device ("transdermal patch"). Examples of suitable
creams, ointments, etc. can be found, for instance, in the
Physician's Desk Reference. Examples of suitable transdermal
devices are described, for instance, in U.S. Pat. No. 4,818,540
issued Apr. 4, 1989 to Chinen, et al., hereby expressly
incorporated by reference in its entirety.
[0472] Compositions that are suitable for parenteral administration
include, but are not limited to, pharmaceutically acceptable
sterile isotonic solutions. Such solutions include, but are not
limited to, saline, phosphate buffered saline and oil preparations
for injection into a central venous line, intravenous,
intramuscular, intraperitoneal, intradermal, or subcutaneous
injection.
[0473] Compositions that are suitable for transbronchial and
transalveolar administration include, but not limited to, various
types of aerosols for inhalation. Devices suitable for
transbronchial and transalveolar administration of these are also
embodiments. Such devices include, but are not limited to,
atomizers and vaporizers. Many forms of currently available
atomizers and vaporizers can be readily adapted to deliver vaccines
having ribavirin and an antigen.
[0474] Compositions that are suitable for gastrointestinal
administration include, but not limited to, pharmaceutically
acceptable powders, pills or liquids for ingestion and
suppositories for rectal administration.
[0475] The nucleic acid constructs described herein, in particular,
may be administered by means including, but not limited to,
traditional syringes, needleless injection devices, or
"microprojectile bombardment gene guns". Alternatively, the genetic
vaccine may be introduced by various means into cells that are
removed from the individual. Such means include, for example, ex
vivo transfection, electroporation, microinjection and
microprojectile bombardment. After the gene construct is taken up
by the cells, they are reimplanted into the individual. It is
contemplated that otherwise non-immunogenic cells that have gene
constructs incorporated therein can be implanted into the
individual even if the vaccinated cells were originally taken from
another individual.
[0476] According to some embodiments, the gene construct is
administered to an individual using a needleless injection device.
In other embodiments, the gene construct comprising the antigen of
interest is provided to an individual in need of an immune response
to said antigen using an electroporation device (e.g., a needle
device or a needleless device). According to some embodiments, the
gene construct is simultaneously administered to an individual
intradermally, subcutaneously and intramuscularly using a
needleless injection device. Needleless injection devices,
multi-needle electroporation devices, and nucleic acid
electroporation devices, in general, are well known and widely
available (See e.g., U.S. Pat. No. 5,273,525, EP 1240917 B1, U.S.
Pat. No. 5,702,359, EP 0874663B1,U.S. Pat. No. 6,418,341, U.S. Pat.
No. 6,763,264 U.S. Pat. No. 6,055,453, U.S. Pat. No. 6,233,482,
U.S. Pat. No. 6,068,650, U.S. Pat. No. 6,014,584, U.S. Pat. No.
6,241,701, U.S. Pat. No. 6,516,223, U.S. Pat. No. 6,678,556, and
U.S. Pat. No. 6,110,161, hereby expressly incorporated by reference
in their entireties).
[0477] One having ordinary skill in the art can, following the
teachings herein, use needleless or needled electroporation devices
(e.g., providing the nucleic acid construct by hypodermic needle
followed by electroporation at the injection site) to deliver
genetic material to cells of an individual. These gene construct
delivery devices are well suited to deliver genetic material to all
tissue. They are particularly useful to deliver genetic material to
skin and muscle cells. In some embodiments, a needleless injection
device may be used to propel a liquid or disolvable substrate or
carrier that comprises the nucleic acid construct (e.g., ballistic
transformation) toward the surface of the individual's skin. The
liquid is propelled at a sufficient velocity such that upon impact
with the skin the liquid penetrates the surface of the skin,
permeates the skin and muscle tissue therebeneath. Thus, the
genetic material is simultaneously administered intradermally,
subcutaneously and intramuscularly. In some embodiments, a
needleless injection device may be used to deliver genetic material
to tissue of other organs in order to introduce a nucleic acid
molecule to cells of that organ.
[0478] Preferred embodiments include methods of enhancing an immune
response to a desired antigen by providing an animal in need with
an amount of adjuvant (e.g., ribavirin) and one or more of the
nucleic acid or polypeptide compositions disclosed herein that is
effective to enhance said immune response. In these embodiments, an
animal in need of an enhanced immune response to an antigen/target
is identified by using currently available diagnostic testing or
clinical evaluation. By one approach, for example, an individual
infected with a virus, or afflicted with cancer, is provided with
the vaccine compositions described above in an amount sufficient to
elicit a cellular and humoral immune response to a viral or cancer
TCE so as to protect said individual from becoming infected with
the virus, or to treat the cancer from which the TCE is derived. In
another embodiment, an individual infected with a virus is
identified and provided with a vaccine composition comprising
ribavirin and either a nucleic acid or polypeptide composition
described herein, that includes a TCE from the virus and NS3/4A
sequences in an amount sufficient to enhance the cellular and
humoral immune response against the viral TCE so as to reduce or
eliminate the viral infection.
[0479] The following Example describes the systematic mutation of
residues in the NS3 protease domain in order to elucidate the
potential insertion sites for TCEs or TCEs and linkers, in which
the chimeric NS3/4A proteins retain protease activity.
Example 14
[0480] The serine protease cleavage domain of NS3/NS4A resides in
the first 181 amino acids of the peptide (Lin, C. et al., J.
Virol., 68(12):8147-8157 (1994)). FIG. 17 depicts the amino acid
sequence of the protease cleavage domain of the NS3/NS4A
polypeptide from HCV isolate disclosed herein (SEQ ID NO: 39). To
identify amino acid residues that affect protease activity in the
HCV isolate, mutations were made in each of the 181 amino acids of
the protease cleavage domain. Briefly, the NS3/NS4A-pVAX plasmid
(described in Example 1) was used as a template for site-directed
mutageneis using the QUICKCHANGE.TM. mutagenesis kit (Stratagene),
following the manufacturer's recommendations. Using this approach,
NS3/NS4A-pVAX constructs encoding the polypeptides of (SEQ ID NO's:
40 through 220 and 1329-1339) were made. These constructs encode
NS3/NS4A polypeptides in which every residue other than alanine in
(SEQ ID NO: 39) is changed to an alanine, and where every alanine
in (SEQ ID NO: 39) is changed to a glycine. The resulting plasmids
were sequenced to verify that the NS3/NS4A-pVAX vectors had been
correctly made. Plasmids were grown in competent BL21 E. coli, and
subsequently purified using Qiagen DNA purification columns
(Qiagen, Hamburg, Germany) according to the manufacturer's
instructions. Purified plasmid DNA was dissolved in phosphate
buffered saline (PBS).
[0481] The resulting plasmids were transcribed and translated in
vitro, and the resulting polypeptides were visualized by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In
vitro transcription and translation were performed using the T7
coupled reticulocyte lysate TNT.TM. system (Promega, Madison, Wis.)
according to the manufacturer's instructions. All in vitro
translation reactions of the constructs were carried out at
30.degree. C. with .sup.35S-labeled methionine (Amersham
International, Plc, Buckinghamshire, UK). The labeled proteins were
separated by 12% SDS-PAGE and visualized by exposure to X-ray film
(Hyper Film-MP, Amersham) for 6-18 hours.
[0482] When the assay described above is performed with
wtNS3/NS4A-pVAX, the protease activity of wtNS3/NS4A protein (SEQ
ID NO: 2) is such that two protein bands are visualized on the
autorad of the gel: a protein band of approximately 67 kDa, which
is consistent with the size of the NS3/NS4A uncleaved protein, and
a protein band of approximately 61 kDa, which corresponds to the
NS3 cleavage product from the reaction. Each of the 181 mutant
NS3/NS4A-pVAX constructs was tested in the assay described above.
For each mutant construct assayed, the amount of uncleaved (67 kDa)
versus cleaved NS3/NS4A (61 kDa cleavage product) was compared
between the wtNS3/NS4A construct and the NS3/NS4A mutant construct,
as a measure of how each mutation affected the protease activity.
As shown in Table 23, the following NS3/NS4A constructs have amino
acid substitutions that completely abolished protease activity: SEQ
ID NOs: 87, 92, 96, 120, 124, 130, 136, 138, 162, 163, 178, 179,
184, 192, 208, and 214. In reference to NS3 protease activity, the
term "completely abolished" is meant to refer to polypeptides that
have less than, equal to, or any number in between about 10%, 9%,
8%, 7%, 6%, 5%, 4%, 3% 2% and 1% of the NS3 protease activity
compared to the protease activity of a wild type NS3 polypeptide or
NS3/4A polyeptide (e.g., SEQ ID NO: 36). In reference to NS3
protease activity, the term "reduced" is meant to refer to
polypeptides that have less than, equal to, or any number in
between about 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%,
50%, 45%, 40%, 35%, 30%, 25%, 20%, 15% and 10% of the NS3 protease
activity compared to the protease activity of a wild type NS3
polypeptide or NS3/4A polyeptide (e.g., SEQ ID NO: 36). The
following eight constructs have mutations that result in reduced
protease activity: SEQ ID NOs: 83, 133, 145, 147, 165, 182, 183,
and 188.
[0483] As shown in Table 24, twenty two constructs have
substitutions that result in enhanced (SEQ ID NOs: 45, 50, 52, 53,
69, 98, 103, 112, 115, 125, 150, 161, 173, 175, 180, 200, 205, and
216), or greatly enhanced protease activity (SEQ ID NOs: 91, 97,
and 197). In reference to NS3 protease activity, the term
"enhanced" and "greatly enhanced" is meant to refer to polypeptides
that have greater than, equal to, or any number in between about
100%, 101%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%,
150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, 200%,
210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%,
320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 425%, 450%,
475%, 500%, 600% and 700% of the NS3 protease activity compared to
the protease activity of a wild type NS3 polypeptide or NS3/4A
polypeptide (e.g., SEQ ID NO: 36).
TABLE-US-00024 TABLE 23 Leu44Ala (SEQ ID NO: 83) Reduced Ile48Ala
(SEQ ID NO: 87) Abolished Trp53Ala (SEQ ID NO: 92) Abolished
His57Ala (SEQ ID NO: 96) Abolished Asp81Ala (SEQ ID NO: 120)
Abolished Trp85Ala (SEQ ID NO: 124) Abolished Ala91Gly (SEQ ID NO:
130) Abolished Leu94Ala (SEQ ID NO: 133) Reduced Cys97Ala (SEQ ID
NO: 136) Abolished Cys99Ala (SEQ ID NO: 138) Abolished Leu106Ala
(SEQ ID NO: 145) Reduced Thr108Ala (SEQ ID NO: 147) Reduced
Arg123Ala (SEQ ID NO: 162) Abolished Gly124Ala (SEQ ID NO: 163)
Abolished Leu126Ala (SEQ ID NO: 165) Reduced Ser139Ala (SEQ ID NO:
178) Abolished Gly140Ala (SEQ ID NO: 179) Abolished Leu143Ala (SEQ
ID NO: 182) Reduced Leu144Ala (SEQ ID NO: 183) Reduced Cys145Ala
(SEQ ID NO: 184) Abolished His149Ala (SEQ ID NO: 188) Reduced
Ile153Ala (SEQ ID NO: 192) Abolished Phe169Ala (SEQ ID NO: 208)
Abolished Leu175Ala (SEQ ID NO: 214) Abolished
TABLE-US-00025 TABLE 24 Mutation Activity Tyr6Ala (SEQ ID NO: 45)
Enhanced Arg11Ala (SEQ ID NO: 50) Enhanced Leu13Ala (SEQ ID NO: 52)
Enhanced Leu14Ala (SED ID NO: 53) Enhanced Glu30Ala (SEQ ID NO: 69)
Enhanced Cys52Ala (SEQ ID NO: 91) Greatly enhanced Gly58Ala (SEQ ID
NO: 97) Greatly enhanced Ala59Gly (SEQ ID NO: 98) Enhanced Ile64Ala
(SEQ ID NO: 103) Enhanced Gln73Ala (SEQ ID NO: 112) Enhanced
Thr76Ala (SEQ ID NO: 115) Enhanced Pro86Ala (SEQ ID NO: 125)
Enhanced Ala111Gly (SEQ ID NO: 150) Enhanced Gly122Ala (SEQ ID NO:
161) Enhanced Tyr134Ala (SEQ ID NO: 173) Enhanced Lys136Ala (SEQ ID
NO: 175) Enhanced Gly141Ala (SEQ ID NO: 180) Enhanced Va1158Ala
(SEQ ID NO: 197) Greatly Enhanced Arg161Ala (SEQ ID NO: 200)
Enhanced Ala166Gly (SEQ ID NO: 205) Enhanced Thr177Ala (SEQ ID NO:
216) Enhanced
[0484] Protease activity is associated with viral assembly and
maturation (See, e.g., Babe et al., Cell, 91:427-430 (1997)).
Accordingly, mutant NS3/NS4A polypeptides with altered protease
activity and their encoding nucleic acids are useful in the
immunogenic compositions described herein. The fragments listed in
TABLES 23-24 are preferred immunogens that can be incorporated with
or without an adjuvant (e.g., ribavirin) into a composition for
administration to an animal so as to induce an immune response in
said animal to HCV.
[0485] As shown in TABLE 25, the following NS3/4A constructs have
amino acid substitutions that did not have a large effect (SEQ ID
NOs: 40, 48-49, 54, 56, 60-61, 66, 72, 74-75, 77-79, 82, 85, 89,
100-102, 107-110, 113-114, 121, 131, 144, 146, 148-149, 152-153,
156, 160, 166, 167, 170-171, 177, 181, 185-186, 189-190, 194-195,
198-199, 204, 206, 209, 210, 213, 215, and 217), or did not have
any detectable effect on protease activity (SEQ ID NOs: 41-44, 46,
47, 51, 55, 58, 59, 62, 65, 67-68, 70-71, 73, 76, 80-81, 84, 86,
88, 90, 93, 95, 99, 104-106, 111, 116-119, 123, 127-129, 134-135,
137, 139-143, 151, 154-155, 157-159, 164, 168-169, 172, 176, 191,
193, 196, 201-203, 211-212, and 218-220). The fragments listed in
TABLE 22 are preferred immunogens that can be incorporated with or
without an adjuvant (e.g., ribavirin) into a composition for
administration to an animal so as to induce an immune response in
said animal to HCV.
TABLE-US-00026 TABLE 25 Mutation Activity Ala1Gly (SEQ ID NO: 40)
Little Effect Pro2Ala (SEQ ID NO: 41) No Effect Ile3Ala (SEQ ID NO:
42) No Effect Thr4Ala (SED ID NO: 43) No Effect Ala5Gly (SEQ ID NO:
44) No Effect Ala7Gly (SEQ ID NO: 46) No Effect Gln8Ala (SEQ ID NO:
47) No Effect Gln9Ala (SEQ ID NO: 48) Little Effect Thr10Ala (SEQ
ID NO: 49) Little Effect Gly12Ala (SEQ ID NO: 51) No Effect
Gly15Ala (SEQ ID NO: 54) Little Effect Cys16Ala (SEQ ID NO: 55) No
Effect Ile17Ala (SEQ ID NO: 56) Little Effect Thr19Ala (SEQ ID NO:
58) No Effect Ser20Ala (SEQ ID NO: 59) No Effect Leu21Ala (SEQ ID
NO: 60) Little Effect Thr22Ala (SEQ ID NO: 61) Little Effect
Gly23Ala (SEQ ID NO: 62) No Effect Lys26Ala (SEQ ID NO: 65) No
Effect Asn27Ala (SEQ ID NO: 66) Little Effect Gln28Ala (SEQ ID NO:
67) No Effect Val29Ala (SEQ ID NO: 68) No Effect Gly31Ala (SEQ ID
NO: 70) No Effect Glu32Ala (SEQ ID NO: 71) No Effect Val33Gly (SEQ
ID NO: 72) Little Effect Gln34Ala (SEQ ID NO: 73) No Effect
Ile35Ala (SEQ ID NO: 74) Little Effect Val36Ala (SEQ ID NO: 75) No
Effect Ser37Ala (SEQ ID NO: 76) Little Effect Thr38Ala (SEQ ID NO:
77) Little Effect Ala39Gly (SEQ ID NO: 78) Little Effect Ala40Gly
(SEQ ID NO: 79) Little Effect Gln41Ala (SEQ ID NO: 80) No Effect
Thr42Ala (SEQ ID NO: 81) No Effect Phe43Ala (SEQ ID NO: 82) Little
Effect Ala45Gly (SEQ ID NO: 84) No Effect Thr46Ala (SEQ ID NO: 85)
Little Effect Cys47Ala (SEQ ID NO: 86) No Effect Gln49Ala (SEQ ID
NO: 88) No Effect Gly50Ala (SEQ ID NO: 89) Little Effect Val51Ala
(SEQ ID NO: 90) Little Effect Thr54Ala (SEQ ID NO: 93) No Effect
Arg161Ala (SEQ ID NO: 95) No Effect Ala56Gly (SEQ ID NO: 99) No
Effect Phe57Ala (SEQ ID NO: 100) Little Effect Leu58Ala (SEQ ID NO:
101) No Effect Thr63Ala (SEQ ID NO: 102) Little Effect Thr64Ala
(SEQ ID NO: 103) No Effect Ala65Gly (SEQ ID NO: 104) No Effect
Ser66Ala (SEQ ID NO: 105) No Effect Pro67Ala (SEQ ID NO: 106) No
Effect Lys68Ala (SEQ ID NO: 107) Little Effect Gly69Ala (SEQ ID NO:
108) Little Effect Pro70Ala (SEQ ID NO: 109) Little Effect Val71Ala
(SEQ ID NO: 110) Little Effect Ile72Ala (SEQ ID NO: 111) Little
Effect Met74Ala (SEQ ID NO: 113) Little Effect Tyr75Ala (SEQ ID NO:
114) Little Effect Gln77Ala (SEQ ID NO: 116) No Effect Val78Ala
(SEQ ID NO: 117) No Effect Asp79Ala (SEQ ID NO: 118) No Effect
Gln80Ala (SEQ ID NO: 119) No Effect Leu82Ala (SEQ ID NO: 121)
Little Effect Gly84Ala (SEQ ID NO: 123) No Effect Pro88Ala (SEQ ID
NO: 127) No Effect Gln89Ala (SEQ ID NO: 128) No Effect Gly90Ala
(SEQ ID NO: 129) No Effect Arg92Ala (SEQ ID NO: 131) Little Effect
Thr95Ala (SEQ ID NO: 134) No Effect Pro96Ala (SEQ ID NO: 135) No
Effect Thr98Ala (SEQ ID NO: 137) No Effect Gly100Ala (SEQ ID NO:
139) No Effect Ser101Ala (SEQ ID NO: 140) No Effect Ser102Ala (SEQ
ID NO: 141) No Effect Asp103Ala (SEQ ID NO: 142) No Effect
Leu104Ala (SEQ ID NO: 143) No Effect Try105Ala (SEQ ID NO: 144)
Little Effect Val107Ala (SEQ ID NO: 146) Little Effect Arg109Ala
(SEQ ID NO: 148) Little Effect His110Ala (SEQ ID NO: 149) Little
Effect Asp112Ala (SEQ ID NO: 151) No Effect Val113Ala (SEQ ID NO:
152) Little Effect Ile114Ala (SEQ ID NO: 153) Little Effect
Proll5Ala (SEQ ID NO: 154) No Effect Va1116Ala (SEQ ID NO: 155) No
Effect Arg118Ala (SEQ ID NO: 157) No Effect Arg119Ala (SEQ ID NO:
158) Little Effect Gly120Ala (SEQ ID NO: 159) No Effect Asp121Ala
(SEQ ID NO: 160) Little Effect Ser125Ala (SEQ ID NO: 164) No Effect
Leu127Ala (SEQ ID NO: 166) Little Effect Ser128Ala (SEQ ID NO: 167)
Little Effect Pro129Ala (SEQ ID NO: 168) No Effect Arg130Ala (SEQ
ID NO: 169) No Effect Pro131Ala (SEQ ID NO: 170) Little Effect
Ile132Ala (SEQ ID NO: 171) Little Effect Ser133Ala (SEQ ID NO: 172)
No Effect Gly137Ala (SEQ ID NO: 176) No Effect Ser138Ala (SEQ ID
NO: 177) Little Effect Pro142Ala (SEQ ID NO: 181) Little Effect
Pro146Ala (SEQ ID NO: 185) Little Effect Ala147Gly (SEQ ID NO: 186)
Little Effect Ala150Gly (SEQ ID NO: 189) Little Effect Val151Gly
(SEQ ID NO: 190) Little Effect Gly152Ala (SEQ ID NO: 191) No Effect
Phe154Ala (SEQ ID NO: 193) No Effect Arg155Ala (SEQ ID NO: 194)
Little Effect Ala156Gly (SEQ ID NO: 195) Little Effect Ala157Gly
(SEQ ID NO: 196) No Effect Cys159Ala (SEQ ID NO: 198) Little Effect
Thr160Ala (SEQ ID NO: 199) Little Effect Gly162Ala (SEQ ID NO: 201)
No Effect Val163Ala (SEQ ID NO: 202) No Effect Ala164Gly (SEQ ID
NO: 203) No Effect Lys165Ala (SEQ ID NO: 204) Little Effect
Val167Ala (SEQ ID NO: 206) Little Effect Ile170Ala (SEQ ID NO: 209)
Little Effect Pro171Ala (SEQ ID NO: 210) Little Effect Val172Ala
(SEQ ID NO: 211) No Effect Glu173Ala (SEQ ID NO: 212) No Effect
Ser174Ala (SEQ ID NO: 213) Little Effect Glu176Ala (SEQ ID NO: 215)
Little Effect Thr178Ala (SEQ ID NO: 217) Little Effect
Met179Ala (SEQ ID NO: 218) No Effect Arg180Ala (SEQ ID NO: 219) No
Effect Ser181Ala (SEQ ID NO: 220) No Effect
[0486] The mutant HCV genes and the encoded polypeptides disclosed
herein are useful as novel research tools for drug discovery.
Specifically, polypeptides exhibiting enhanced protease activity
can be used in assays to identify novel compounds that inhibit
protease activity. Such screening assays will include assays
amenable to high-throughput screening of chemical libraries, making
them particularly suitable for identifying small molecule drug
candidates. Small molecules contemplated include synthetic organic
or inorganic compounds. The assays can be performed in a variety of
formats, including protein-protein binding assays, biochemical
screening assays, immunoassays and cell based assays, which are
well characterized in the art.
[0487] The HCV genes encoding polypeptides with altered protease
activity are useful in the creation of transgenic organisms, as
described herein in paragraphs [0183]-[0184]. Transgenic organisms
expressing mutant HCV polypeptides are useful as model organisms
for the study of HCV replication and life cycle.
Example 15
[0488] Two particular mutants, Val1055Ala (corresponding to
Val29Ala of SEQ ID NO: 68) and Gln1060Ala (corresponding to
Gln34Ala of SEQ ID NO: 73), were tested for their ability to
proteolytically cleave the NS3-NS4A junction while not cleaving the
human IPS-1 molecule to .DELTA.IPS-1. As detailed in above,
Val29Ala of SEQ ID NO: 68 and Gln34Ala of SEQ ID NO: 73 were tested
for their ability to affect the protease activity in the HCV
isolate. These mutants were found through mutagenesis of the NS3
protease domain wherein the residues depicted in FIG. 18 were
replaced with Alanine or Glycine and tested for their effect on
cleavage of the NS3-NS4A cleavage site (also depicted in FIG. 18).
Also mentioned above, these two mutants did not have a large effect
on protease activity in the HCV isolate. These mutants were further
tested for their ability to cleave the human IPS-1 molecule to
.DELTA.IPS-1.
[0489] Cells were cotransfected with a plasmid coding for the human
IPS-1 gene as well as a pVAX1 plasmid expressing the mutant NS3/4A
gene. 36 hours later the cells were lyse. Cleaved and uncleaved
IPS-1 peptides were visualized on an SDS-PAGE gel after a Western
blot with antibodies specific for the IPS-1 fragments. As shown in
FIGS. 19A and 19B Val1055Ala (corresponding to Val29Ala of SEQ ID
NO: 68) and Gln1060Ala (corresponding to Gln34Ala of SEQ ID NO: 73)
were not able to cleave IPS-1 to .DELTA.IPS-1.
Example 16
[0490] A particular mutant, containing two amino acid mutations
Val1055Ala and and Gln1060Ala (corresponding to Val29Ala and
Gln34Ala, respectively, of SEQ ID NO: 1329), is created by
combining the mutations present in SEQ ID NOs.: 68 and 73. The
residues referred to as 1055 and 1060 vary by 1026 amino acids from
the listed sequences as the NS3/4A gene begins at residue 1026 of
the HCV polyprotein. This mutant is tested for its affect on the
proteolytic cleavage of the NS3-NS4A junction as in Example 15.
Results show that the mutant does not have a large effect on
protease activity in the HCV isolate.
[0491] This mutant is further tested for its ability to cleave the
human IPS-1 molecule to .DELTA.IPS-1 as detailed above. Results
show that the IPS-1 gene is not cleaved to .DELTA.IPS-1 by the
mutant represented by SEQ ID NO: 1329.
Example 17
[0492] NS3 protease mutants represented by SEQ ID NOs: 1330-1339
are tested for their ability to affect NS3 protease cleavage at the
NS3-NS4A protease cleavage site, as explained in above. Mutants
having no effect, little effect, no substantial effect, or
heightened effect on protease cleavage at the NS3-NS4A protease
cleavage site are selected for further testing.
[0493] These mutants are further tested for their ability to cleave
the human IPS-1 molecule to .DELTA.IPS-1 as detailed above. Mutants
that cannot cleave IPS-1 to .DELTA.IPS-1 are selected as favorable
mutants.
Example 18
[0494] Mutants containing two or more amino acid mutations, created
by combining any number of favorable mutants described above, are
created. These mutants are tested for their ability to affect NS3
protease cleavage at the NS3-NS4A protease cleavage site, as
detailed in above. Mutants having no effect, little effect, no
substantial effect, or heightened effect on protease cleavage at
the NS3-NS4A protease cleavage site are selected for further
testing.
[0495] These mutants are further tested for their ability to cleave
the human IPS-1 molecule to .DELTA.IPS-1 as detailed above. Mutants
that cannot cleave IPS-1 to .DELTA.IPS-1 are selected as favorable
mutants.
[0496] Although the invention has been described with reference to
embodiments and examples, it should be understood that various
modifications can be made without departing from the spirit of the
invention. Accordingly, the invention is limited only by the
following claims.
[0497] The next example demonstrates that chimeric NS3/4A nucleic
acids and encoded polypeptides described herein prime CTL responses
to the T cell epitopes encoded therein.
Example 19
[0498] The Hepatitis B viral core protein (HBc) has been disclosed
as an immunogenic moiety that stimulates the T cell response of an
immunized host animal. See, e.g, U.S. Pat. No. 4,818,527, U.S. Pat.
No. 4,882,145 and U.S. Pat. No. 5,143,726. More particularly, the
sequence of SEQ ID NO: 1014 of the Hepatitis B core protein has
been shown to elicit a specific T-cell response when administered
to mice. To assess the ability of SEQ ID NO: 1014 DNA constructs to
prime CTLs, the nucleic acid of SEQ ID NO: 1015 is cloned into the
pVAX1 expression vector (Invitrogen, Carlsbad, Calif.) to create
HBcAg-pVAX1.
[0499] Plasmids are grown in BL21 E. coli cells, and sequenced for
accuracy. Plasmid DNA used for in vivo vaccination is purified
using Qiagen DNA purification columns, according to the
manufacturer's instructions (Qiagen GmbH, Hilden, FRG). The
concentration of the resulting plasmid DNA is determined
spectrophotometrically (Dynaquant, Pharmacia Biotech, Uppsala,
Sweden) and the purified DNA is dissolved in sterile phosphate
buffered saline (PBS) at a concentration of 1 mg/ml.
[0500] Groups of eight to ten C57/BL6 mice are primed with
HBcAg-pVAX1 intra muscularly (i.m.) or using a gene gun. For i.m.
delivery, mice are immunized by needle injections of 100 .mu.g
plasmid DNA given intramuscularly to the tibialis anterior (TA)
muscle. 5 days prior to DNA immunization, mice are injected
intramuscularly with 50 .mu.l per TA muscle of 0.01 mM cardiotoxin
(Latoxan) in 0/9% sterile saline. The mice are boosted with a
second injection of 100 .mu.g plasmid DNA four weeks subsequent to
the first DNA immunization. For gene gun delivery, plasmid DNA is
linked to gold particles according to protocols supplied by the
manufacturer (Bio-Rad Laboratories, Hercules, Calif.). Prior to
immunization, the injection area is shaved and the immunization is
performed according to the manufacturer's protocol. Each injection
dose contains 4 .mu.g of plasmid DNA. Immunizations are performed
on weeks 0 and 4.
[0501] The presence of CTLs specific for SEQ ID NO: 1014 is assayed
using a standard .sup.51Cr-release assay. Briefly, spleen cells are
harvested from mice 14 days after the initial immunization or the
booster immunization. Chromium release assays are performed as
described in Lazdina, et al. (2003) J. Gen. Virol. 84:1-8, herein
expressly incorporated by reference in its entirety. Single cell
suspensions are prepared. 25.times.10.sup.6 splenocytes are
restimulated with 25.times.10.sup.6 syngenic irradiated (20 Gy)
spelnocytes pulsed with 0.05 .mu.M peptide, as previously
described. Sandberg et al. (2000) J. Immunol. 165:25-33, herein
expressly incorporated by reference in its entirety. Restimulation
cultures are set in 12 ml complete RPMI medium (Gibco). After 5
days, effector cells are harvested and washed twice. RMA-S target
cells (Karre et al. (1986) Nature 319:675-678) are pulsed with 50
.mu.M peptide for 90 min at 5% CO.sub.2 and 37.degree. C. Serial
dilutions of effector cells are incubated with 5.times.103
chromium-labeled peptide pulsed RMA-S target cells in a final
volume of 200 .mu.l per well in 96-well plates. After a 4 hour
incubation at 5% CO.sub.2 and 37.degree. C., 100 .mu.l of
supernatant is collected and the radioactivity is determined using
a .gamma. counter. The percentage of specific release is calculated
according to the formula: (Experimental release-spontaneous
release/total release-spontaneous release).times.100.
[0502] The results of the .sup.51Cr-release assay is shown in FIG.
20A. i.m. injection of HBcAg-pVAX1 elicits a cellular immune
response. By contrast, immunization with HBcAg-pVAX1 via a gene gun
does not elicit a cellular immune response. FIG. 20B.
[0503] In another set of experiments, the presence of CTLs specific
for the SEQ ID NO: 1014 is assayed using a standard ELISPOT assay
to detect .gamma.-IFN-secreting CTLs. Current Protocols in
Immunology, Edited by: John E. Coligan, Ada M. Kruisbeek, David H.
Margulies, Ethan M. Shevach, Warren Strober (2001 John Wiley &
Sons, NY, N.Y.)
[0504] In still another set of experiments, the NS3/4A chimeric
polypeptides encoded by the nucleic acids are used to immunize
mice, using standard immunization procedures for polypeptides such
as those disclosed in co-pending U.S. Patent Application No.
60/685,014, the contents of which is hereby expressly incorporated
by reference in its entirety.
[0505] In contrast to HBcAg-pVAX1, nucleic acids encoding the
NS3/4A peptide can effectively prime CTLs by both intra muscular
and gene gun delivery. See, e.g., co-pending U.S. Provisional
Patent Application No. 60/685,014. To demonstrate that NS3/4A
functions as a T-cell epitope carrier, chimeric NS3/4A nucleic
acids that include the TCE encoded by SEQ ID NO: 1015, or the
encoded polypeptides, are tested for their ability to prime CTLs by
both i.m. and gene gun delivery. The NS3/4A-pVAX vector described
in Example 1 is used to generate NS3/4A chimeric expression vectors
containing in-frame fusions of SEQ ID NO: 1015 using standard
cloning techniques. See, Ausubel et al., supra. The chimeric NS3/4A
expression vectors contain in-frame fusions of SEQ ID NO: 1015 to
the 5' end of the NS3/4A coding sequence; to the 3' end of the
NS3/4A coding sequence, and within the NS3/4A coding sequence such
that the epitope of SEQ ID NO: 1015 is between amino acids 181 and
182 of SEQ ID NO: 36, between amino acid residues 453 and 513 of
SEQ ID NO: 36 (e.g., SEQ ID NO: 1013, which encodes the NS3/4A
chimeric polypeptide of SEQ ID NO: 1012) or in analogous positions
in any NS3/4A polypeptide, or elsewhere within the NS3/4A
polypeptide. The chimeric NS3/4A nucleic acids are delivered to
mice either intramuscularly or using a gene gun, as described
herein. Specific CTL responses are measured using a
.sup.51Cr-release assay or ELIspot assay.
[0506] The ability of the chimeric NS3/4A vectors to prime CTLs is
similar whether the vector is administered intramuscularly or using
a gene gun, demonstrating that presentation of epitopes in the
context of NS3/4A effectively primes CTLs against the epitopes.
This example also suggests that the NS3/4A platform is useful for
generating immune responses to HBV TCEs that elicit immune
responses in humans, (e.g., SEQ ID SEQ ID NO: 351).
[0507] The following example describes the generation and
validation of immunogenic compositions that generate or enhance CTL
priming to specific antigens.
Example 20
[0508] Chimeric NS3/4A nucleic acid constructs encoding at least
one TCE juxtaposed to or inserted within various positions along
the NS3/4A polypeptide are made and assayed for their ability to
prime an immune response to the TCE. Chimeric polypeptides encoded
by the NS3/4A chimeric nucleic acids are also assayed for their
ability to prime an immune response to the encoded TCE. A TCE to
which a CTL response is desired (e.g., any one of the TCEs
presented herein, including SEQ ID NOs: 221-271, SEQ ID NOs:
809-1011, and SEQ ID NO: 1014) is selected. Using standard cloning
techniques, the nucleic acid encoding the TCE (e.g., any one of the
TCEs presented herein, including SEQ ID NOs: 221-271, SEQ ID NOs:
809-1011, and SEQ ID NO: 1014) is cloned into the NS3/4A-pVAX
vector described in Example 1, or an equivalent thereof (e.g., an
NS3/4A-pVAX vector wherein the NS3/4A sequence is selected from the
group of SEQ ID NOs: 572-808) to generate a chimeric NS3/4A-pVAX
vector. The chimeric NS3/4A-pVAX vectors encode chimeric NS3/4A
polypeptides in which the TCE is juxtaposed to the N-terminus or
C-terminus of the NS3/4A polypeptide, or is located within the
NS3/4A polypeptide (e.g., between amino acids 181 and 182 of SEQ ID
NO: 2).
[0509] Plasmids that have been sequenced for accuracy are purified
and prepared for use in immunization as described in Example 19.
Alternatively, polypeptides encoded by said nucleic acids are
expressed and used in immunizations as described in Example 19.
Mice are primed with the chimeric NS3/4A-pVAX nucleic acids intra
muscularly (i.m.) or using a gene gun as described in Example 19,
or by another method (e.g., using electroporation (Innovio, Oslo,
Sweden) according to the manufacturer's instructions).
[0510] The priming of CTLs specific for the TCE (e.g., an epitope
listed in presented herein, including SEQ ID NOs: 221-271, SEQ ID
NOs: 809-1011, and SEQ ID NO:1014) is assayed using a standard
.sup.51Cr-release assay or a standard ELISPOT assay to detect
.gamma.-IFN-secreting CTLs. Data from the .sup.51Cr-release assay
or the ELISPOT assay are used to determine preferred sites of
insertion of the TCE within the NS3/4A-pVAX vector
[0511] Chimeric NS3/4A expression vectors contain in-frame fusions
of TCEs (e.g., an epitope listed in presented herein, including SEQ
ID NOs: 221-271, SEQ ID NOs: 809-1011, and SEQ ID NO: 1014) to the
5' end of the NS3/4A coding sequence; to the 3' end of the NS3/4A
coding sequence, and within the NS3/4A coding sequence such that
the TCE is between amino acids 181 and 182 of SEQ ID NO: 36,
between amino acid residues 453 and 513 of SEQ ID NO: 36, or in
analogous positions in any NS3/4A polypeptide, or elsewhere within
the NS3/4A polypeptide. The chimeric NS3/4A nucleic acids or
encoded polypeptides are delivered to mice either intramuscularly
or using a gene gun, as described herein. Specific CTL responses
are measured using a .sup.51Cr-release assay or ELISPOT assay as
described in Example 3.
[0512] For each TCE, preferred sites of insertion within an NS3/4A
nucleic acid, or juxtaposed to the NS3/4A nucleic acid are
determined by comparing the immune responses generated by the
chimeric nucleic acids or encoded polypeptides. Accordingly,
provided herein are methods of making an immunogen that can include
the steps of a) identifying a TCE against which an immune response
is desired b) generating at least one chimeric NS3/4A nucleic acid
in which the DNA sequence encoding the TCE is juxtaposed to or
inserted within the NS3/4A sequence (e.g., SEQ ID NO: 1), and c)
detecting the immune response generated by the chimeric NS3/4A
nucleic acid or encoded polypeptide.
Example 21
[0513] The Hepatitis B viral core protein (HBc) is an immunogen
that stimulates the T cell response of an immunized host animal.
See, e.g, U.S. Pat. No. 4,818,527, U.S. Pat. No. 4,882,145 and U.S.
Pat. No. 5,143,726, all of which are hereby expressly incorporated
by reference in their entireties. In fact, the Hepatitis B core
protein (HBcAg) has been shown to elicit a specific T-cell response
in immunized mice. It is contemplated that DNA immunogens that are
codon-optimized for expression in humans and which encode the HCV
NS3/4A platform and fragments of HBcAg separated by NS3 protease
cleavage sites will effectively prime HBcAg-specific CTLs,
stimulate HBcAg-specific proliferative T cell responses, and induce
production of HBcAg-specific antibodies in animals when these DNA
immunogens are delivered by various DNA vaccination methodologies.
In some embodiments, it is contemplated that the DNA immunogens,
which are codon-optimized for expression in humans and which encode
the HCV NS3/4A platform and fragments of HBcAg separated by NS3
protease cleavage sites will be more effective at priming
HBcAg-specific CTLs, stimulating HBcAg-specific proliferative T
cell responses, and inducing production of HBcAg-specific
antibodies in animals than conventional DNA immunogens that encode
HBcAg antigens and more effective than DNA immunogens that encode
the NS3/4A platform and fragments of HBcAg without NS3 protease
cleavage sites.
[0514] To determine the immunogenicity of codon-optimized DNA
constructs encoding the HCV NS3/4A platform and fragments of HBcAg
separated by NS3 protease cleavage sites and to compare the
efficiency of these constructs with conventional HBcAg-containing
constructs with and without the NS3/4A platform, several
codon-optimized DNA constructs encoding the HCV NS3/4A platform and
fragments of HBcAg separated by NS3 protease cleavage sites
including antigenic sequences in various orientations are made (see
SEQ ID NOs: 1174-1198 and FIG. 1). Codon-optimized DNA constructs
encoding only the HBcAg and/or fragments thereof or encoding the
NS3/4A platform and the HBcAg and/or fragments thereof without NS3
protease cleavage sites are also made for comparison. Codon
optimized DNA encoding the HCV NS3/4A platform and fragments of
HBcAg in various orientations separated by NS3 protease cleavage
sites are cloned into the pVAX1 expression vector (Invitrogen,
Carlsbad, Calif.) or other suitable DNA vaccination vectors. Once
the constructs are made, they are provided to animals by a DNA
vaccination methodology (e.g., injection, electroporation, such as
MedPulser.RTM., or intranasal or transdermal delivery). Analysis of
the presence and amount of HBcAg-specific CTLs can then be made
before during and after several introductions of the constructs
(e.g., an initial introduction followed by one, two, three, four,
or five boosting events). It will be shown that the presence of the
HCV NS3/4A platform provides a more robust DNA immunogen, as
compared to immunogens that lack the NS3/4A platform, and that the
presence of one or more NS3/4A protease cleavage sites within the
antigen also improves immunogenicity. It is also expected that the
presence of shuffled HBcAg antigenic fragments (e.g., SEQ ID NOs:
1191-1198) within the antigen will provide a greater immune
response than the unshuffled native antigen or fragments thereof.
The following describes these experiments in greater detail.
[0515] Plasmids containing the codon-optimized (human) DNA
immunogens encoding the HCV NS3/4A platform and fragments of HBcAg
separated by the NS3/4A protease cleavage site will be grown in
BL21 E. coli cells, and sequenced for accuracy. Although the
fragments of HBcAg are separated by the NS3/4A protease cleavage
site, any NS3 protease cleavage site can be used (e.g., NS4A/B,
NS4B/5A, and NS5A/B). The NS3/4A platform is separated from the
fragments of HBcAg by an NS4A/B cleavage site, although any NS3
protease cleavage site can be used. In the construct, the NS3
.mu.latform is separated from the NS4A by the NS3/4A protease
cleavage site, although any NS3 protease cleavage site can be used.
Plasmids containing the conventional HBcAg sequence and/or
fragments thereof will also be grown for comparison. Plasmid DNA
used for in vivo vaccination is then purified using Qiagen DNA
purification columns, according to the manufacturer's instructions
(Qiagen GmbH, Hilden, FRG). The concentration of the resulting
plasmid DNA is determined spectrophotometrically (Dynaquant,
Pharmacia Biotech, Uppsala, Sweden) and the purified DNA is
dissolved in sterile phosphate buffered saline (PBS) at a
concentration of approximately 1 mg/ml.
[0516] Groups of eight to ten C57/BL6 mice or New Zealand rabbits
are primed with an HBcAg-containing construct (see SEQ ID NOs:
1174-1198 and FIG. 1) intranasally, transdermally, intra muscularly
(i.m.), or using an electroporation device (e.g.,
MedPulser.RTM.).
[0517] If a transdermal or intranasal delivery is evaluated, an
amount of plasmid DNA that is sufficient to deliver approximately
70 .mu.g-100 .mu.g of plasmid DNA per dose is formulated with the
delivery vehicle. Animals are then provided the plasmid DNA one,
two, three, four, or five times at monthly intervals. Prior to
transdermal immunization, the delivery area is shaved.
[0518] If intramuscular injection is evaluated, animals are
immunized i.m with approximately 70-100 .mu.g plasmid DNA at the
tibialis anterior (TA) muscle. 5 days prior to DNA immunization,
animals may also be injected intramuscularly with 50 .mu.l per TA
muscle of 0.01 mM cardiotoxin (Latoxan) in 0/9% sterile saline.
[0519] When electroporation is evaluated, animals are immunized i.m
with approximately 70-100 .mu.g plasmid DNA at the tibialis
anterior (TA) muscle and immediately after injection, the
Medpulser.RTM. is applied with a 0.5 cm needle array set to deliver
two 60 ms pulses of 246 V/cm to the injection site. In mice, one
two needle electrode tip is used and when rabbits are used, one
four needle electrode tip is used per injection per animal. The
procedure can be repeated up to three times in mice and up to five
times in rabbits at monthly intervals.
[0520] If gene gun delivery is performed, plasmid DNA is linked to
gold particles according to protocols supplied by the manufacturer
(Bio-Rad Laboratories, Hercules, Calif.). Prior to immunization,
the injection area is shaved and the immunization is performed
according to the manufacturer's protocol. Each injection dose by
gene gun contains 4-100 .mu.g of plasmid DNA. Immunizations are
performed on weeks 0 and 4.
[0521] The presence of CTLs specific for HBcAg is then assayed
using a standard .sup.51Cr-release assay. Briefly, spleen cells are
harvested from immunized animals 14 days after the initial
immunization or a booster immunization. Chromium release assays are
performed as described in Lazdina, et al. (2003) J. Gen. Virol.
84:1-8, herein expressly incorporated by reference in its entirety.
Single cell suspensions are prepared. 25.times.10.sup.6 splenocytes
are restimulated with 25.times.10.sup.6 syngenic irradiated (20 Gy)
spelnocytes pulsed with 0.05 .mu.M peptide, as previously
described. Sandberg et al. (2000) J. Immunol. 165:25-33, herein
expressly incorporated by reference in its entirety. Restimulation
cultures are set in 12 ml complete RPMI medium (Gibco). After 5
days, effector cells are harvested and washed twice. RMA-S target
cells (Karre et al. (1986) Nature 319:675-678) are pulsed with 5
.mu.M peptide for 90 min at 5% CO.sub.2 and 37.degree. C. Serial
dilutions of effector cells are incubated with 5.times.10.sup.3
chromium-labeled peptide pulsed RMA-S target cells in a final
volume of 200 .mu.l per well in 96-well plates. After a 4 hour
incubation at 5% CO.sub.2 and 37.degree. C., 100 .mu.l of
supernatant is collected and the radioactivity is determined using
a .gamma. counter. The percentage of specific release is calculated
according to the formula: (Experimental release-spontaneous
release/total release-spontaneous release).times.100. The results
of the .sup.51Cr-release assay will show that the presence of the
HCV NS3/4A platform provides a more robust DNA immunogen, as
compared to immunogens that lack the NS3/4A platform, and that the
presence of one or more NS3/4A protease cleavage sites within the
antigen also improves immunogenicity. The assay will further show
that the presence of shuffled HBcAg antigenic fragments (e.g., SEQ
ID NOs: 1191-1198) within the antigen will provide a greater immune
response than the unshuffled native antigen or fragments
thereof.
[0522] In another set of experiments, the presence of
.gamma.-IFN-secreting CTLs and T helper (Th) cells to HBcAg in
spleenocyte or lymph node cultures will be evaluated using a
commercially available ELISpot assay. (See Current Protocols in
Immunology, Edited by: John E. Coligan, Ada M. Kruisbeek, David H.
Margulies, Ethan M. Shevach, Warren Strober (2001 John Wiley &
Sons, NY, N.Y.), herein expressly incorporated by reference in its
entirety). By this approach, the number of .gamma.-IFN-secreting
CTLs or spots is determined at various concentrations of peptide.
These experiments will show that the presence of the HCV NS3/4A
platform provides a more robust DNA immunogen, as compared to
immunogens that lack the NS3/4A platform, and that the presence of
one or more NS3/4A protease cleavage sites within the antigen also
improves immunogenicity. The assay will further show that the
presence of shuffled HBcAg antigenic fragments (e.g., SEQ ID NOs:
1191-1198) within the antigen will provide a greater immune
response than the unshuffled native antigen or fragments
thereof.
[0523] In still another set of experiments, the proliferative
responses to HbcAg in whole blood obtained from immunized animals
is determined. An amount of whole blood is obtained from the animal
(e.g., approximately 4 ml from a rabbit), prior to the first
vaccination and two weeks after each vaccination. The blood is
collected in Heparin tubes and the plasma and peripheral
mononuclear cells (PBMCs) are isolated by gradient centrifugation.
The plasma is stored at -80 degrees Centrigrade until analysis for
HBcAg-specific antibodies. The presence and amount of antibodies
specific for HBcAg in the various samples can be measured using an
ELISA assay. The PBMCs are immediately assayed for in vitro
proliferative recall responses using a standard 96 h proliferation
assay. (See Lazinda et al., J. Gen. Virol. 82:1299-1308 (2001),
herein expressly incorporated by reference in its entirety.) In
brief, microtiter plates are seeded with approximately 200,000
cells/well and the cells are incubated with media alone,
phytohemagglutinin (PHA) or recombinant HbcAg. After 72 hours,
radioactive thymidine is added and 16-24 hours later the cells are
harvested, The proliferation is determined as radioactivity of the
cells as the counts per minute (cpm) of cells incubated with the
antigen divided by the CPM of the cells incubated with the media
alone (sample to negative ration; S/N). Groups are compared by the
mean S/N ratios at several time points). These experiments will
show that the presence of the HCV NS3/4A platform provides a more
robust DNA immunogen, as compared to immunogens that lack the
NS3/4A platform, and that the presence of one or more NS3/4A
protease cleavage sites within the antigen also improves
immunogenicity. The assay will also show that the presence of
shuffled HBcAg antigenic fragments (e.g., SEQ ID NOs: 1191-1198)
within the antigen will provide a greater immune response than the
unshuffled native antigen or fragments thereof.
[0524] In yet another set of experiments, tumor inhibition assays
will be carried out. Two weeks after the last immunization, mice
will be challenged using tumor cells expressing the corresponding
vaccine antigen, and protection against tumor growth will be
measured. These experiments will show that the presence of the HCV
NS3/4A platform provides a more robust DNA immunogen, as compared
to immunogens that lack the NS3/4A platform, and that the presence
of one or more NS3/4A protease cleavage sites within the antigen
also improves immunogenicity. The assay will also show that the
presence of shuffled HBcAg antigenic fragments (e.g., SEQ ID NOs:
1191-1198) within the antigen will provide a greater immune
response than the unshuffled native antigen or fragments
thereof.
[0525] In still another set of experiments, quantification of HBcAg
CTL responses will be measured by flow cytometry (Tetramers and
Dimer-X). These experiments will show that the presence of the HCV
NS3/4A platform provides a more robust DNA immunogen, as compared
to immunogens that lack the NS3/4A platform, and that the presence
of one or more NS3/4A protease cleavage sites within the antigen
also improves immunogenicity. The assay will also show that the
presence of shuffled HBcAg antigenic fragments (e.g., SEQ ID NOs:
1191-1198) within the antigen will provide a greater immune
response than the unshuffled native antigen or fragments
thereof.
Example 22
[0526] A similar methodology as that provided in EXAMPLE 21 can be
applied to evaluate any DNA immunogen provided herein. More
specifically, it is contemplated that DNA immunogens that are
codon-optimized for expression in humans and which encode the HCV
NS3/4A platform and one or more fragments of the antigens provided
in SEQ ID NOs: 1016-1034, SEQ ID NOs: 1146-1173 and SEQ ID NOs:
1210-1328, wherein said fragments are separated by NS3 protease
cleavage sites will effectively prime antigen-specific CTLs,
stimulate antigen-specific proliferative T cell responses, and
induce production of antigen-specific antibodies in animals when
these DNA immunogens are delivered by various DNA vaccination
methodologies. Examples of antigen fragments of SEQ ID NOs:
1019-1021, SEQ ID NO: 1146, SEQ ID NOs: 1150-1166, SEQ ID NO: 1168,
SEQ ID NO: 1170, and SEQ ID NO: 1172 separated by the NS3 protease
cleavage site NS3/4A are presented inSEQ ID NOs: 1122-1145.
Although the fragments in SEQ ID NOs: 1122-1145 are separated by
the NS3/4A protease cleavage site, any NS3 protease cleavage site
can be used (e.g., NS4A/B, NS4B/5A, and NS5A/B). Additionally, it
is contemplated that DNA immunogens that are codon-optimized for
expression in humans and which encode the HCV NS3/4A platform and a
plurality of antigenic fragments from the antigens presented in SEQ
ID NOs: 1016-1034, SEQ ID NOs: 1146-1173 and SEQ ID NOs: 1210-1328,
separated by NS3 protease cleavage sites, including antigenic
sequences in various orientations as seen with the HBcAg from
earlier examples, will also effectively prime antigen-specific
CTLs, stimulate antigen-specific proliferative T cell responses,
and induce production of antigen-specific antibodies in animals
when these DNA immunogens are delivered by various DNA vaccination
methodologies. In some embodiments, it is contemplated that the DNA
immunogens, which are codon-optimized for expression in humans and
which encode the HCV NS3/4A platform and one or more fragments of
the antigens provided in SEQ ID NOs: 1016-1034, SEQ ID NOs:
1146-1173 and SEQ ID NOs: 1210-1328 separated by a NS3 protease
cleavage site will be more effective at priming antigen-specific
CTLs, stimulating antigen-specific proliferative T cell responses,
and inducing production of antigen-specific antibodies in animals
than conventional DNA immunogens that encode the antigens
alone.
[0527] DNA constructs encoding the HCV NS3/4A platform and
fragments of the antigens presented in SEQ ID NOs: 1019-1021, SEQ
ID NOs: 1146-1173 and SEQ ID NOs: 1210-1328 separated by NS3
protease cleavage sites, including antigenic sequences in various
orientations, are made. SEQ ID NOs: 1098-1121 presents codon
optimized fragments of antigens presented in SEQ ID NOs: 1019-1021,
SEQ ID NO: 1146, SEQ ID NOs: 1150-1166, SEQ ID NO: 1168, SEQ ID NO:
1170, and SEQ ID NO: 1172, wherein the fragments are separated by
NS3/4A protease cleavage sites. Although the fragments are
separated by NS3/4A protease cleavage sites, any NS3 protease
cleavage site can be used. Additionally, although the fragments
presented in SEQ ID NOs: 1098-1121 are configured in a naturally
occurring order, separated by NS3 protease cleavage sites,
fragments in various orientations, similar to the shuffled
fragments of HBcAg in SEQ ID NOs: 1191-1198, are made. The shuffled
fragments of the antigens presented in SEQ ID NOs: 1019-1021, SEQ
ID NO: 1146, SEQ ID NOs: 1150-1166, SEQ ID NO: 1168, SEQ ID NO:
1170, and SEQ ID NO: 1172 are also separated by an NS3 protease
cleavage site. Codon-optimized DNA constructs encoding only the
fragments of antigen presented in SEQ ID NOs: 1016-1034, SEQ ID
NOs: 1146-1173 and SEQ ID NOs: 1210-1328 or encoding the NS3/4A
platform and the fragments of antigen presented in SEQ ID NOs:
1016-1034, SEQ ID NOs: 1146-1173 and SEQ ID NOs: 1210-1328 without
NS3 protease cleavage sites are also made for comparison. Codon
optimized DNA encoding the HCV NS3/4A platform and fragments of
antigen presented in SEQ ID NOs: 1016-1034, SEQ ID NOs: 1146-1173
and SEQ ID NOs: 1210-1328 in various orientations separated by NS3
protease cleavage sites are cloned into the pVAX1 expression vector
(Invitrogen, Carlsbad, Calif.) or other suitable DNA vaccination
vectors. Once the constructs are made, they are provided to animals
by a DNA vaccination methodology (e.g., injection, electroporation,
such as MedPulser.RTM., or intranasal or transdermal delivery).
Analysis of the presence and amount of antigen-specific CTLs can
then be made before during and after several introductions of the
constructs (e.g., an initial introduction followed by one, two,
three, four, or five boosting events). It will be shown that the
presence of the HCV NS3/4A platform provides a more robust DNA
immunogen, as compared to immunogens that lack the NS3/4A platform,
and that the presence of one or more NS3/4A protease cleavage sites
within the antigen also improves immunogenicity. It is also
expected that the presence of shuffled antigenic fragments within
the antigen will provide a greater immune response than the
unshuffled native antigen or fragments thereof. The following
describes these experiments in greater detail.
[0528] Plasmids containing the codon-optimized (human) DNA
immunogens encoding the HCV NS3/4A platform and fragments of
antigen presented in SEQ ID NOs: 1016-1034, SEQ ID NOs: 1146-1173
and SEQ ID NOs: 1210-1328 separated by the NS3/4A protease cleavage
site will be grown in BL21 E. coli cells, and sequenced for
accuracy. Although the fragments of antigen presented in SEQ ID
NOs: 1016-1034, SEQ ID NOs: 1146-1173 and SEQ ID NOs: 1210-1328 are
separated by the NS3/4A protease cleavage site, any NS3 protease
cleavage site can be used (e.g., NS4A/B, NS4B/5A, and NS5A/B). The
NS3/4A platform is separated from the fragments of antigen
presented in SEQ ID NOs: 1016-1034, SEQ ID NOs: 1146-1173 and SEQ
ID NOs: 1210-1328 by an NS4A/B cleavage site, although any NS3
protease cleavage site can be used. In the construct, the NS3
.mu.latform is separated from the NS4A by the NS3/4A protease
cleavage site, although any NS3 protease cleavage site can be used.
Plasmids containing codon-optimized nucleic acids encoding
conventional fragments of antigen presented in SEQ ID NOs:
1016-1034, SEQ ID NOs: 1146-1173 and SEQ ID NOs: 1210-1328 will
also be grown for comparison. Plasmid DNA used for in vivo
vaccination is then purified using Qiagen DNA purification columns,
according to the manufacturer's instructions (Qiagen GmbH, Hilden,
FRG). The concentration of the resulting plasmid DNA is determined
spectrophotometrically (Dynaquant, Pharmacia Biotech, Uppsala,
Sweden) and the purified DNA is dissolved in sterile phosphate
buffered saline (PBS) at a concentration of approximately 1
mg/ml.
[0529] Groups of eight to ten C57/BL6 mice or New Zealand rabbits
are primed with an antigen-containing construct intranasally,
transdermally, intra muscularly (i.m.), or using an electroporation
device (e.g., MedPulser.RTM.).
[0530] If a transdermal or intranasal delivery is evaluated, an
amount of plasmid DNA that is sufficient to deliver approximately
70 .mu.g-100 .mu.g of plasmid DNA per dose is formulated with the
delivery vehicle. Animals are then provided the plasmid DNA one,
two, three, four, or five times at monthly intervals. Prior to
transdermal immunization, the delivery area is shaved.
[0531] If intramuscular injection is evaluated, animals are
immunized i.m with approximately 70-100 .mu.g plasmid DNA at the
tibialis anterior (TA) muscle. 5 days prior to DNA immunization,
animals may also be injected intramuscularly with 50 .mu.l per TA
muscle of 0.01 mM cardiotoxin (Latoxan) in 0/9% sterile saline.
[0532] When electroporation is evaluated, animals are immunized i.m
with approximately 70-100 .mu.g plasmid DNA at the tibialis
anterior (TA) muscle and immediately after injection, the
Medpulser.RTM. is applied with a 0.5 cm needle array set to deliver
two 60 ms pulses of 246 V/cm to the injection site. In mice, one
two needle electrode tip is used and when rabbits are used, one
four needle electrode tip is used per injection per animal. The
procedure can be repeated up to three times in mice and up to five
times in rabbits at monthly intervals.
[0533] If gene gun delivery is performed, plasmid DNA is linked to
gold particles according to protocols supplied by the manufacturer
(Bio-Rad Laboratories, Hercules, Calif.). Prior to immunization,
the injection area is shaved and the immunization is performed
according to the manufacturer's protocol. Each injection dose by
gene gun contains 4-100 g of plasmid DNA. Immunizations are
performed on weeks 0 and 4.
[0534] The presence of CTLs specific for antigen is then assayed
using a standard .sup.51Cr-release assay. Briefly, spleen cells are
harvested from immunized animals 14 days after the initial
immunization or a booster immunization. Chromium release assays are
performed as described in Lazdina, et al. (2003) J. Gen. Virol.
84:1-8, herein expressly incorporated by reference in its entirety.
Single cell suspensions are prepared. 25.times.10.sup.6 splenocytes
are restimulated with 25.times.10.sup.6 syngenic irradiated (20 Gy)
spelnocytes pulsed with 0.05 .mu.M peptide, as previously
described. Sandberg et al. (2000) J. Immunol. 165:25-33, herein
expressly incorporated by reference in its entirety. Restimulation
cultures are set in 12 ml complete RPMI medium (Gibco). After 5
days, effector cells are harvested and washed twice. RMA-S target
cells (Karre et al. (1986) Nature 319:675-678) are pulsed with 50
.mu.M peptide for 90 min at 5% CO.sub.2 and 37.degree. C. Serial
dilutions of effector cells are incubated with 5.times.10.sup.3
chromium-labeled peptide pulsed RMA-S target cells in a final
volume of 200 .mu.l per well in 96-well plates. After a 4 hour
incubation at 5% CO.sub.2 and 37.degree. C., 100 .mu.l of
supernatant is collected and the radioactivity is determined using
a .gamma. counter. The percentage of specific release is calculated
according to the formula: (Experimental release-spontaneous
release/total release-spontaneous release).times.100. The results
of the .sup.51Cr-release assay will show that the presence of the
HCV NS3/4A platform provides a more robust DNA immunogen, as
compared to immunogens that lack the NS3/4A platform, and that the
presence of one or more NS3/4A protease cleavage sites within the
antigen also improves immunogenicity. The assay will further show
that the presence of shuffled antigenic fragments within the
antigen will provide a greater immune response than the unshuffled
native antigen or fragments thereof.
[0535] In another set of experiments, the presence of
.gamma.-IFN-secreting CTLs and T helper (Th) cells to antigens in
spleenocyte or lymph node cultures will be evaluated using a
commercially available ELISpot assay. (See Current Protocols in
Immunology, Edited by: John E. Coligan, Ada M. Kruisbeek, David H.
Margulies, Ethan M. Shevach, Warren Strober (2001 John Wiley &
Sons, NY, N.Y.), herein expressly incorporated by reference in its
entirety). By this approach, the number of .gamma.-IFN-secreting
CTLs or spots is determined at various concentrations of peptide.
These experiments will show that the presence of the HCV NS3/4A
platform provides a more robust DNA immunogen, as compared to
immunogens that lack the NS3/4A platform, and that the presence of
one or more NS3/4A protease cleavage sites within the antigen also
improves immunogenicity. The assay will further show that the
presence of shuffled antigenic fragments within the antigen will
provide a greater immune response than the unshuffled native
antigen or fragments thereof.
[0536] In still another set of experiments, the proliferative
responses to antigen in whole blood obtained from immunized animals
is determined. An amount of whole blood is obtained from the animal
(e.g., approximately 4 ml from a rabbit), prior to the first
vaccination and two weeks after each vaccination. The blood is
collected in Heparin tubes and the plasma and peripheral
mononuclear cells (PBMCs) are isolated by gradient centrifugation.
The plasma is stored at -80 degrees Centrigrade until analysis for
antigen-specific antibodies. The presence and amount of antibodies
specific for antigen in the various samples can be measured using
an ELISA assay. The PBMCs are immediately assayed for in vitro
proliferative recall responses using a standard 96 h proliferation
assay. (See Lazinda et al., J. Gen. Virol. 82:1299-1308 (2001),
herein expressly incorporated by reference in its entirety.) In
brief, microtiter plates are seeded with approximately 200,000
cells/well and the cells are incubated with media alone,
phytohemagglutinin (PHA) or recombinant antigen. After 72 hours,
radioactive thymidine is added and 16-24 hours later the cells are
harvested, The proliferation is determined as radioactivity of the
cells as the counts per minute (cpm) of cells incubated with the
antigen divided by the CPM of the cells incubated with the media
alone (sample to negative ration; S/N). Groups are compared by the
mean S/N ratios at several time points). These experiments assay
will show that the presence of the HCV NS3/4A platform provides a
more robust DNA immunogen, as compared to immunogens that lack the
NS3/4A platform, and that the presence of one or more NS3/4A
protease cleavage sites within the antigen also improves
immunogenicity. The assay will further show that the presence of
shuffled antigenic fragments within the antigen will provide a
greater immune response than the unshuffled native antigen or
fragments thereof.
[0537] In yet another set of experiments, tumor inhibition assays
will be carried out. Two weeks after the last immunization, mice
will be challenged using tumor cells expressing the corresponding
vaccine antigen, and protection against tumor growth will be
measured. These experiments will show that the presence of the HCV
NS3/4A platform provides a more robust DNA immunogen, as compared
to immunogens that lack the NS3/4A platform, and that the presence
of one or more NS3/4A protease cleavage sites within the antigen
also improves immunogenicity. The assay will further show that the
presence of shuffled antigenic fragments within the antigen will
provide a greater immune response than the unshuffled native
antigen or fragments thereof.
[0538] In still another set of experiments, quantification of
antigen CTL responses will be measured by flow cytometry (Tetramer
and Dimer-X). These experiments assay will show that the presence
of the HCV NS3/4A platform provides a more robust DNA immunogen, as
compared to immunogens that lack the NS3/4A platform, and that the
presence of one or more NS3/4A protease cleavage sites within the
antigen also improves immunogenicity. The assay will further show
that the presence of shuffled antigenic fragments within the
antigen will provide a greater immune response than the unshuffled
native antigen or fragments thereof.
[0539] Although the invention has been described with reference to
embodiments and examples, it should be understood that various
modification can be made without departing from the spirit of the
invention. Accordingly, the invention is limited only by the
following claims. All of the patents, patent applications, and
references cited herein are expressly incorporated by reference in
their entireties.
Example 23
[0540] Groups of C57/BL6 mice were immunized twice with 50 .mu.g of
either plasmid containing a codon optimized NS3/4A gene, a
NS3/4A-Betv1 fusion gene containing a protease cleavage site
between NS3 and NS4A as well as a protease cleavage site between
the NS4A and the birch antigen (SEQ ID NO: 1380), or a NS3/4A-Betv1
fusion gene containing a protease cleavage site between NS3 and
NS4A, a protease cleavage site between the NS4A and the birch
antigen as well as two additional protease cleavage sites within
the birch antigen (SEQ ID NO: 1381) using an electroporation
device. Another group of mice were immunized twice with recombinant
Betv1 protein (rBetv1) in Freunds incomplete adjuvant. The two
immunizations were 4 weeks apart. The mice were sacrificed two
weeks after the second immunizations and the lymph nodes and
spleens of each group were collected and analyzed.
[0541] The presence of .gamma.-IFN-secreting CTLs and T helper (Th)
cells to antigens in spleenocyte or lymph node cultures were
evaluated using a commercially available ELISpot assay. (See
Current Protocols in Immunology, Edited by: John E. Coligan, Ada M.
Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober
(2001 John Wiley & Sons, NY, N.Y.), herein expressly
incorporated by reference in its entirety). The spleen and lymph
nodes from each group were pooled and immediately tested for the
presence of NS3 and birch specific T cells. The ability of
NS3-specific and birch-specific Th and CTLs to produce .gamma.-IFN
recalled by a concanavalin-A (con-A) as a positive control, media
alone as a negative control, native HBcAg, recombinant HBcAg
(rHBcAg), various concentrations of NS3/4A CTL peptide, various
concentrations of rNS3, and various concentrations of rBetv1 was
analyzed via the ELISpot. The results of the ELISpot assay are
shown in FIG. 22.
[0542] The NS3/4A-Betv1 (major Birch allergen) fusion genes showed
that the NS3/4A clearly functions as an adjuvant for IFN-.gamma.
production. The NS3/4A-Betv1 fusion genes showed that the liberated
NS3/4A-Betv1 fusion gene (SEQ ID NO: 1380) and fragmented
NS3/4A-Betv1 fusion gene (SEQ ID NO: 1381) effective prime
IFN-.gamma. producing Betv1-specific T cells two weeks after the
second injection, where the recombinant Betv1 antigen fails to do.
The data suggests the fragmented NS3/4A birch antigen fusion gene
(SEQ ID NO: 1381) more effectively primes IFN-.gamma. producing
T-cells than the non-fragmented NS3/4A birch antigen fusion gene
(SEQ ID NO: 1380).
Example 24
[0543] Groups of C57/BL6 mice were immunized twice with 50 .mu.g of
either plasmid containing a codon optimized NS3/4A gene, a
NS3/4A-Betv1 fusion gene containing a protease cleavage site
between NS3 and NS4A as well as a protease cleavage site between
the NS4A and the birch antigen (SEQ ID NO: 1380), or a NS3/4A-Betv1
fusion gene containing a protease cleavage site between NS3 and
NS4A, a protease cleavage site between the NS4A and the birch
antigen as well as two additional protease cleavage sites within
the birch antigen (SEQ ID NO: 1381) using an electroporation
device. Another group of mice were immunized twice with recombinant
Betv1 protein (rBetv1) in Freunds incomplete adjuvant. The two
immunizations were 4 weeks apart. The mice were bled two weeks
after the second immunizations.
[0544] Dilutions of the sera were tested on ELISA plates coated
with recombinant birch protein. A secondary antibody specific for
IgE antibodies was used to detect bound antibody. The results are
shown in FIG. 23. The results show that when looking at the priming
of IgE to Betv1, the DNA constructs do not prime IgE antibodies
whereas the rBetv1 shows birch-specific IgE antibodies. The DNA
constructs are thus not allergenic in that they prime a Th1 type
response.
Example 25
[0545] Groups of C57/BL6 mice were immunized twice with 50 .mu.g
using an electroporation device with either plasmid containing:
[0546] a plasmid encoding a codon optimized NS3/4A gene, [0547] a
naked pVAX-1 plasmid [0548] a plasmid containing a gene encoding
HBcAg [0549] a plasmid containing an NS3/4A-HBcAg fusion gene as
follows: [0550] C1 (SEQ ID NO: 1382) having an active protease but
no protease cleavage site anywhere on the fusion gene [0551] C2
(SEQ ID NO: 1383) having an inactive protease and no protease
cleavage site anywhere on the fusion gene [0552] C3 (SEQ ID NO:
1384) having an active protease and a protease cleavage site
between NS3 and NS4A but no protease cleavage site anywhere else on
the fusion gene [0553] C4 (SEQ ID NO: 1385) having an active
protease and a protease cleavage site between NS3 and NS4A and a
protease cleavage site between NS4A and HBcAg [0554] C5 (SEQ ID NO:
1386) having an active protease and a protease cleavage site
between NS3 and NS4A a protease cleavage site between NS4A and
HBcAg and 3 protease cleavage sites within the HBcAg which is in a
naturally occurring order [0555] C5 (SEQ ID NO: 1386) having an
active protease and a protease cleavage site between NS3 and NS4A a
protease cleavage site between NS4A and HBcAg and 3 protease
cleavage sites within the HBcAg which is in a naturally occurring
order [0556] C6 (SEQ ID NO: 1387) having an active protease and a
protease cleavage site between NS3 and NS4A a protease cleavage
site between NS4A and HBcAg and 3 protease cleavage sites within
the HBcAg which is in a non-naturally occurring order
[0557] The two immunizations were 4 weeks apart. The mice were
sacrificed two weeks after the second immunizations and the lymph
nodes and spleen from each mouse was collected.
[0558] The presence of CTLs specific for antigen was then assayed
using a standard .sup.51Cr-release assay. Briefly, the collected
cells were harvested from immunized animals 14 days after the
booster immunization. Chromium release assays were performed as
described in Lazdina, et al. (2003) J. Gen. Virol. 84:1-8, herein
expressly incorporated by reference in its entirety. Single cell
suspensions are prepared. 25.times.10.sup.6 splenocytes were
restimulated with 25.times.10.sup.6 syngenic irradiated (20 Gy)
spelnocytes pulsed with 0.05 .mu.M peptide, as previously
described. Sandberg et al. (2000) J. Immunol. 165:25-33, herein
expressly incorporated by reference in its entirety. Restimulation
cultures were set in 12 ml complete RPMI medium (Gibco). After 5
days, effector cells were harvested and washed twice. RMA-S target
cells (Karre et al. (1986) Nature 319:675-678) were pulsed with 50
.mu.M peptide for 90 min at 5% CO.sub.2 and 37.degree. C. Serial
dilutions of effector cells were incubated with 5.times.10.sup.3
chromium-labeled peptide pulsed RMA-S target cells in a final
volume of 200 .mu.l per well in 96-well plates. After a 4 hour
incubation at 5% CO.sub.2 and 37.degree. C., 100 .mu.l of
supernatant was collected and the radioactivity was determined
using a .gamma. counter. The percentage of specific release was
calculated according to the formula: (Experimental
release-spontaneous release/total release-spontaneous
release).times.100. The results of the .sup.51Cr-release assay is
presented in FIG. 24. NS3/4A clearly functions as an effective
adjuvant for lytic CTL priming. The peak CTL levels for the
non-fragmented HBcAg were higher compared to all other constructs.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20170058003A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20170058003A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References