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United States Patent |
6,180,768
|
Maertens
,   et al.
|
January 30, 2001
|
Sequences of hepatitis C virus genotypes and their use as prophylactic,
therapeutic and diagnostic agents
Abstract
The present invention relates to new genomic nucleotide sequences and amino
acid sequences corresponding to the coding region of these genomes. The
invention relates to new HCV types and subtypes sequences which are
different from the known HCV types and subtypes sequences. More
particularly, the present invention relates to new HCV type 7 sequences,
new HCV type 9 sequences, new HCV type 10 and new HCV type 11 sequences.
Also, the present invention relates to new HCV type 1 sequences of
subtypes 1d, 1e, 1f and 1g; new HCV type 2 sequences of subtypes 2e, 2f,
2g, 2h, 2i, 2k and 2l; new HCV type 3 sequences of subtype 3g, new HCV
type 4 sequences of subtypes 4k, 4l and 4m; a process for preparing them,
and their use for diagnosis, prophylaxis and therapy. More particularly,
the present invention provides new type-specific sequences of the Core,
the E1 and NS5 regions of new HCV types 7, 9, 10 and 11, as well as of new
variants (subtypes) of HCV types 1, 2, 3 and 4. These new HCV sequences
are useful to diagnose the presence of HCV type 1, and/or type 2, and/or
type 3, and/or type 4, and/or type 7, and/or type 9, and/or type 10,
and/or type 11 genotypes or serotypes in a biological sample. Moreover,
the availability of these new type-specific sequences can increase the
overall sensitivity of HCV detection and should also prove to be useful
for prophylactic and therapeutic purposes.
Inventors:
|
Maertens; Geert (Bruges, BE);
Stuyver; Lieven (Herzele, BE)
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Assignee:
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Innogenetics N.V. (Ghent, BE)
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Appl. No.:
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836075 |
Filed:
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April 21, 1997 |
PCT Filed:
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October 23, 1995
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PCT NO:
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PCT/EP95/04155
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371 Date:
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April 21, 1997
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102(e) Date:
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April 21, 1997
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PCT PUB.NO.:
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WO96/13590 |
PCT PUB. Date:
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May 9, 1996 |
Foreign Application Priority Data
| Oct 21, 1994[EP] | 94870166 |
| Jun 28, 1995[EP] | 95870076 |
Intern'l Class: |
C07H 021/04; C12Q 001/70; G01N 033/53; C12P 021/00 |
Field of Search: |
435/5,7.1,320.1,69.3,252.3
530/300,350
536/23.1,23.72,24.3
|
References Cited
Foreign Patent Documents |
6-319563 | Nov., 1994 | JP.
| |
93-00365 | Jan., 1993 | WO.
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94-25601 | Nov., 1994 | WO.
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Other References
Liu et al. Jan. 1992 Gene 114 (2) 245-250.
Stuyver et al. 194 Journal of General Virology 74 (6) 1093-1102, Jan. 1994.
Stuyver et al. Jan. 1994 PNAS USA 91 (21) 10134-10138.
van Doorn et al, Jan. 1994 Journal of Hepatology 21 (1) 122-129.
Qu et al. May 1994 Journal of General Virology 75 (5)0 1063-1070.
|
Primary Examiner: Eisenschenk; Frank C.
Assistant Examiner: Zeman; Mary K
Attorney, Agent or Firm: Arnold, White & Durkee
Claims
We claim:
1. A Hepatitis C virus polynucleic acid, having a nucleotide sequence which
is unique to at least one of the new HCV types 7, 9, 10 or 11, or, to at
least one of the subtypes 1d, 1e, 1f, 1g, 2e, 2f, 2g, 2h, 2i, 2k, 2l, 3g,
4k, 4l or 4m, wherein when the sequence is unique to at least subtype 1d
the sequence is at least 96% identical to SEQ ID NO: 1; or the complement
thereof.
2. A polynucleic acid which is chosen from the group consisting of
(i) the nucleotide sequences having SEQ ID 1, 3, 5, 7, 9, 11, 13 15, 17,
19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53,
55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89,
91, 93, 95, 97, 99, 101, 103 or 105,
(ii) a part of said polynucleic acid of (i) which is unique to at least one
of the new HCV types 7, 9, 10 or 11, or, to at least one of the subtypes
1d, 1e, 1f, 1g, 2e, 2f, 2g, 2h, 2i, 2k, 2l, 3g, 4k, 4l or 4m, wherein when
the sequence is unique to at least subtype 1d the sequence is at least 96%
identical to SEQ ID NO: 1, and
(iii) the complement of the polynucleic acid of (i) or (ii).
3. A polynucleic acid according to claim 1, wherein the polynucleic acid is
selected from
(i) a polynucleic acid encoding an HCV polyprotein comprising in its amino
acid sequence at least one of the following amino acid residues; I15, C38,
V44, A49, Q43, P49, Q55, A58, S60 or D60, E68 or V68, H70, A71 or Q71 or
N71, D72, H81, H101, D106, S110, L130, I134, E135, L140, S148, T150 or
E150, Q153, F155, D157, G160, E165, I169, F181, L186, T190, T192 or I192
or H192, I193, A195, S196, R197 or N197 or K197, Q199 or D199 or H199 or
N199, F200 or T200, A208, I213, M216 or S216, N217 or S217 or G217 or
K217, T218, I219, A222, Y223, I230, W231 or L231, S232 or H232 or A232,
Q233, E235 or L235, F236 or 6236, F237, L240 or M240, A242, N244, N249,
I250 or K250 or R250, A252 or C252, A254, I255 or V255, D256 or M256,
E257, E260 or K260, R261, V268, S272 or R272, I285, G290 or F290, A291,
A293 or L293 or W293, T294 or A294, S295 or H295, K296 or 3296, Y297 or
M297, I299 or Y299, I300, S301, P316, S2646, A2648, G2649, A2650, V2652,
Q2653, H2656 or L2656, F2659, K2663 or 12663, A2667 or V1667, D2677,
L2681, M2686 or Q2686 or E2686, A2692 or K2692, H2697, I2707, L2708 or
Y2708, A2709, A2719 or M2719, F2727, T2728 or D2728, E2729, F2730 or
72730, I2745, V2746 or E2746 or L2746 or K2746, A2748, S2749 or P2749,
R2750, E2751, D2752 or N2752 or S2752 or T2752 or V2752 or I2752 or Q2752,
S2753 or D2753 or G2753, D2754, A2755, L2756 or Q2756, R2757, with said
notation being composed of a letter representing the amino acid residue by
its one-letter code, and a number representing the amino acid numbering as
shown in Table 1,
(ii) a part of said polynucleic acid of (i) which is unique to at least one
of the new HCV types 7, 9, 10 or 11, or, to at least one of the subtypes
1d, 1e, 1f, 1g, 2e, 2f, 2g, 2h, 2i, 2k, 2l, 3g, 4k, 4l or 4m, wherein when
the sequence is unique to at least subtype 1d the sequence is at least 96%
identical to SEQ ID NO: 1,
(iii) or the complement of the polynucleic acid of (i) or (ii).
4. A polynucleic acid according to claim 1, wherein the polynucleic acid is
selected from
(i) a polynucleic acid encoding an HCV polyprotein comprising in its amino
acid sequences at least one amino acid sequence chosen from the group
consisting of the amino acid sequences having SEQ ID 2, 4, 6, 8, 10, 12,
14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 40, 42, 44, 46, 48, 50, 52,
54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 78, 80, 82, 84, 86,
88, 90, 92, 94, 96, 98, 100, 102, 104 or 106,
(ii) a part of said polynucleic acid of (i) which is unique to at least one
of the new HCV types 7, 9, 10 or 11, or, to at least one of the subtypes
1d, 1e, 1f, 1g, 2e, 2f, 2g, 2h, 2i, 2k, 2l, 3g, 4k, 4l or 4m, wherein when
the sequence is unique to at least subtype 1d the sequence is at least 96%
identical to SEQ ID NO: 1,
(iii) or the complement of the polynucleic acid of (i) or (ii).
5. A polynucleic acid according to claim 1, wherein the polynucleic acid is
selected from
(i) a polynucleic acid encoding an HCV polyprotein comprising in its amino
acid sequence at least one amino acid sequence chosen from the group
consisting of the amino acid sequences having SEQ ID 107 to 207,
(ii) a part of said polynucleic acid of (i) which is unique to at least one
of the new HCV types 7, 9, 10 or 11, or, to at least one of the subtypes
1d, 1e, 1f, 1g, 2e, 2f, 2g, 2h, 2i, 2k, 2l, 3g, 4k, 4l or 4m, wherein when
the sequence is unique to at least subtype 1d the sequence is at least 96%
identical to SEQ ID NO: 1,
(iii) or the complement of the polynucleic acid of (ii) or (iii).
6. A polynucleic acid according to any of claims 1 to 5 which comprises 5'
UR sequences, the Core/E1 and the NS4 or the NS5B region or a part
thereof.
7. A recombinant polypeptide encoded by a polynucleic acid according to any
of claims 1 to 5, or a part thereof which is unique to at least one of the
new HCV types 7, 9, 10 or 11, or, to at least one of the subtypes 1d, 1e,
1f, 1g, 2e, 2f, 2g, 2h, 2i, 2k, 2l, 3g, 4k, 4l or 4m, wherein when the
sequence is unique to at least subtype 1d the sequence is at least 96%
identical to SEQ ID NO: 1.
8. A method for production of a recombinant polypeptide, comprising:
transformation of an appropriate cellular host with a recombinant vector,
in which a polynucleic acid or a part thereof according to any of claims 1
to 5 has been inserted under the control of the appropriate regulatory
elements, the polynucleic acid or the part thereof thus being an insert,
culturing said transformed cellular host under conditions enabling the
expression of said insert, and
harvesting said polypeptide.
9. A recombinant expression vector comprising a polynucleic acid or a part
thereof according to any of claims 1 to 5 operably linked to prokaryotic,
eukaryotic or viral transcription and translation control elements.
10. A host cell transformed with a recombinant vector according to claim 9.
11. A peptide corresponding to an amino acid sequence encoded by one of the
polynucleic acids according to any of claims 1 to 5, with said peptide
comprising an epitope which is unique to at least one of the new HCV types
7, 9, 10 or 11, or, to at least one of the subtypes 1d, 1e, 1f, 1g, 2e,
2f, 2g, 2h, 2i, 2k, 2l, 3g, 4k, 4l or 4m, wherein when the sequence is
unique to at least subtype 1d the sequence is at least 96% identical to
SEQ ID NO: 1.
12. The polynucleic acid of claim 1, wherein when the sequence is unique to
at least subtype 1d the sequence is at least 99% identical to SEQ ID NO:1.
Description
The invention relates to new sequences of hepatitis C virus (HCV) genotypes
and their use as prophylactic, therapeutic and diagnostic agents.
The present invention relates to new genomic nucleotide sequences and amino
acid sequences corresponding to the coding region of these genomes. The
invention relates to new HCV types and subtypes sequences which are
different from the known HCV types and subtypes sequences. More
particularly, the present invention relates to new HCV type 7 sequences,
new HCV type 9 sequences, new HCV types 10 and new HCV type 11 sequences.
Also the present invention relates to new HCV type 1 sequences of subtypes
1d, 1e, 1f and 1g; new HCV type 2 sequences of subtypes 2e, 2f, 2g, 2h,
2i, 2k and 2l; new HCV type 3 sequences of subtype 3g, new HCV type 4
sequences of subtypes 4k, 4l and 4m; a process for preparing them, and
their use for diagnosis, prophylaxis and therapy.
The technical problem underlying the present invention is to provide new
HCV sequences from until now unknown HCV types and/or subtypes. More
particularly, the present invention provides new type-specific sequences
of the Core, the E1 and the NS5 regions of new HCV types 7, 9, 10 and 11,
as well as of new variants (subtypes) of HCV types 1, 2, 3 and 4. These
new HCV sequences are useful to diagnose the presence of HCV type 1,
and/or type 2, and/or type 3, and/or type 4, and/or type 7, and/or type 9,
and/or type 10, and/or type 11 genotypes or serotypes in a biological
sample. Moreover, the availability of these new type-specific sequences
can increase the overall sensitivity of HCV detection and should also
prove to be useful for prophylactic and therapeutic purposes.
Hepatitis C viruses (HCV) have been found to be the major cause of non-A,
non-B hepatitis. The sequences of cDNA clones covering the complete genome
of several prototype isolates have been determined (Kato et al., 1990;
Choo et al., 1991; Okamoto et al., 1991; Okamoto et al., 1992). Comparison
of these isolates shows that the variability in nucleotide sequences can
be used to distinguish at least 2 different genotypes, type 1 (HCV-1 and
HCV-J) and type 2 (HC-J6 and HC-J8), with an average homology of about
68%. Within each type, at least two subtypes exist (e.g. represented by
HCV-1 and HCV-J), having an average homology of about 79%. HCV genomes
belonging to the same subtype show average homologies of more than 90%
(Okamoto et al., 1992). However, the partial nucleotide sequence of the
NS5 region of the HCV-T isolates showed at most 67% homology with the
previously published sequences, indicating the existence of yet another
HCV type (Mori et al., 1992). Parts of the 5' untranslated region (UR),
core, NS3, and NS5 regions of this type 3 have been published, further
establishing the similar evolutionary distances between the 3 major
genotypes and their subtypes (Chan et al., 1992). Type 4 was subsequently
discovered (Stuyver et al., 1993b; Simmonds et al., 1993a; Bukh et al.,
1993; Stuyver et al., 1994a). As well as type 5 (Stuyver et al., 1993b;
Simmonds et al., 1993c; Bukh et al., 1993; Stuyver et al., 1994b), and
type 6 HCV groups (Bukh et al., 1993; Simmonds et al., 1993c). An overview
of the present state of the art regarding HCV genotypes is given in Table
3. The nomenclature system proposed by the inventors of the present
application has now been accepted by scientists worldwide (Simmonds et
al., 1994).
The aim of the present invention is to provide new HCV nucleotide and amino
acid sequences enabling the detection of HCV infection.
Another aim of the present infection is to provide new nucleotide and amino
acid HCV sequences enabling the classification of infected biological
fluids into different serological groups.
Another aim of the present invention is to provide new nucleotide and amino
acid HCV sequences ameliorating the overall HCV detection rate.
Another aim of the present invention is to provide new HCV sequences,
useful for the design of HCV prophylactic or therapeutic vaccine
compositions.
Another aim of the present invention is to provide a pharmaceutical
composition consisting of antibodies raised against the polypeptides
encoded by these new HCV sequences, for therapy or diagnosis.
All the aims of the present invention are met by the following embodiments
of the present invention.
The present invention relates more particularly to an HCV polynucleic acid,
having a nucleotide sequence which is unique to a heretofore unidentified
HCV type or subtype which is different from HCV subtypes 1a, 1b, 1c, 2a,
2b, 2c, 2d, 3a, 3b, 3c, 3d, 3e, 3f, 4a, 4b, 4c, 4d, 4e, 4f, 4g, 4h, 4i,
4j, 5a or 6a, with said HCV subtypes being classified as in Table 3 by
comparison of a part of the NS5 gene nucleotide sequence spanning
positions 7932 to 8271, with said amino acid numbering being shown in
Table 1, and with said polynucleic acid containing at least one nucleotide
differing from said known HCV nucleotide sequences, or the complement
thereof. The sequence of known HCV isolates may be found in any nucleotide
sequence database known in the art (such as for instance the EMBL
database).
The present invention thus also relates to a polynucleic acid having a
nucleotide sequence which is unique to at least one of HCV subtypes 1d,
1e, 1f, 1g, 2e, 2f, 2g, 2h, 2i, 2k, 2l, 3g, 4k, 4l, 4m, 7a, 7c or 7d, with
said HCV being classified as defined above.
The present invention thus also relates to a polynucleic acid having a
nucleotide sequence which is unique to at least one of HCV types 9, 10 or
11, with said HCV types being classified as defined above.
It is to be noted that the nucleotide(s) difference in the polynucleic
acids of the invention may involve an amino acid difference in the
corresponding amino acid sequences encoded by said polynucleic acids. A
composition according to the present invention may contain only
polynucleic acid sequences or polynucleic acid sequences mixed with any
excipient known in the art of diagnosis, prophylaxis or therapy.
According to a preferred embodiment, the present invention relates to a
polynucleic acid encoding an HCV polyprotein comprising in its amino acid
sequence at least one of the following amino acid residues:
I15, C38, V44, A49, Q43, P49, Q55, A58, S60 or D60, E68 or V68, H70, A71 or
Q71 or N71, D72, H81, H101, D106, S110, L130, I134, E135, L140, S148, T150
or E150, Q153, F155, D157, G160, E165, I169, F181, L186, T190, T192 or
I192 or H192, I193, A195, S196, R197 or N197 or K197, Q199 or D199 or H199
or N199, F200 or T200, A208, I213, M216 or S216, N217 or S217 or G217 or
K217, T218, I219, A222, Y223, I230, W231 or L231, S232 or H232 or A232,
Q233, E235 or L235, F236 or T236, F237, L240 or M240, A242, N244, N249,
I250 or K250 or R250, A252 or C252, A254, I255 or V255, D256 or M256,
E257, E260 or K260, R261, V268, S272 or R272, I285, G290 or F290, A291,
A293 or L293 or W293, T294 or A294, S295 or H295, K296 or E296, Y297 or
M297, I299 or Y299, I300, S301, P316, S2646, A2648, G2649, A2650, V2652,
Q2653, H2656 or L2656, D2657, F2659, K2663 or Q2663, A2667 or V1667,
D2677, L2681, M2686 or Q2686 or E2686, A2692 or K2692, H2697, I2707, L2708
or Y2708, A2709, A2719 or M2719, F2727, T2728 or D2728, E2729, F2730 or
Y2730, I2741, I2745, V2746 or E2746 or L2746 or K2746, A2748, S2749 or
P2749, R2750, E2751, D2752 or N2752 or S2752 or T2752 or V2752 or I2752 or
Q2752, S2753 or D2753 or G2753, D2754, A2755, L2756 or Q2756, R2757,
with said notation being composed of a letter representing the amino acid
residue by its one-letter code, and a number representing the amino acid
numbering according to Kato et al. (1980), as shown in Table 1, or a part
of said polynucleic acid which is unique to at least one of the HCV
subtypes or types as defined in Table 5, and which contains at least one
nucleotide differing from known HCV nucleotide sequences, or the
complement thereof.
Each of the above-mentioned residues can be found in FIGS. 2, 4 or 6
showing the new amino acid sequences of the present invention aligned with
known sequences of other types or subtypes of HCV for the Core/E1 region.
According to another preferred embodiment, the present invention relates to
a polynucleic acid encoding a HCV polyprotein comprising in its amino acid
sequence at least one amino acid sequence chosen from the following list:
ARQSDGRSWAQ or ARRSEGRSWAQ as for subtype 1d (SEQ ID NO 107 and 108)
ERRPEGRSWAQ as for subtype 1e (SEQ ID NO 109)
ARRPEGRSWAQ as for subtype 1f (SEQ ID NO 110)
DRRTTGKSWGR as for subtype 2k (SEQ ID NO 111)
DRRATGRSWGR as for subtype 2e (SEQ ID NO 112)
DRRATGKSWGR as for subtype 2f (SEQ ID NO 113)
VRQPTGRSWGQ as for type 9 (SEQ ID NO 114)
VRHQTGRTWAQ as for subtype 7a and 7c (SEQ ID NO 115)
VRQNQGRTWAQ as for subtype 7d (SEQ ID NO 116)
ARRTEGRSWAQ as for type 10 (SEQ ID NO 117)
VRRTTGRXXXX or VRRTTGRTWAQ as for type 11 (SEQ ID NO 118 and 119)
HEVRNASGVYHV or HEVRNASGVYHL as for subtype 1d (SEQ ID NO 120 and 121)
YEVHSTTDGYHV as for subtype 1f (SEQ ID NO 122)
VEVKNTSQAYMA as for subtype 2e (SEQ ID NO 123)
IQVKNNSHFYMA as for subtype 2f (SEQ ID NO 124)
VQVKNTSTMYMA as for subtype 2g (SEQ ID NO 125)
VQVKNTSHSYMV as for subtype 2h (SEQ ID NO 126)
VQVANRSGSYMV as for subtype 2i (SEQ ID NO 127)
VEIKNTXNTYVL or VEIKNTSNTYVL as for subtype 2k (SEQ ID NO 128 and 129)
INYRNVSGIYYV or INYRNTSGIYHV or INYHNTSGIYHI or TNYRNVSGIYHV as for subtype
4k (SEQ ID NO 130, 131, 132 or 133)
QHYRNVSGIYHV as for subtype 4l (SEQ ID NO 134)
IQVKNASGIYHL as for type 9 (SEQ ID NO 135)
AHYTNKSGLYHL as for subtype 7c (SEQ ID NO 136)
LNYANKSGLYHL as for subtype 7d (SEQ ID NO 137)
LEYRNASGLYMV as for type 10 (SEQ ID NO 138)
IYEMDGMIMHY or IYEMSGMILHA as for subtype 1d (SEQ ID NO 139 and 140)
VYEAKDIILHT as for subtype 1f (SEQ ID NO 141)
VWQLXDAVLHV as for subtype 2e (SEQ ID NO 142)
VWQLRDAVLHV as for subtype 2f (SEQ ID NO 143)
IWQMQGAVLHV as for subtype 2g (SEQ ID NO 144)
VWQLKDAVLHV as for subtype 2h (SEQ ID NO 145)
VWQLEEAVLHV as for subtype 2i (SEQ ID NO 146)
TWQLXXAVLHV as for subtype 2k (SEQ ID NO 147)
VYEADHHILHL or VYEADHHILAL or VFEADHHILHL as for subtupe 4k (SEQ ID NO 148,
149 and 150)
VYESDHHILHL as for subtype 4l (SEQ ID NO 151)
VFEAETMILHL as for type 9 (SEQ ID NO 152)
VYEAETLILHL as for subtype 7c (SEQ ID NO 153)
VYEANGMILHL as for subtype 7d (SEQ ID NO 154)
VYEAGDIILHL as for type 10 (SEQ ID NO 155)
VREDNHLRCWMAL or VRENNSSRCWMAL as for subtype 1d (SEQ ID NO 156 and 157)
IREGNISRCWVPL as for subtype 1f (SEQ ID NO 158)
ENSSGRFHCWIPI as for subtype 2e (SEQ ID NO 159)
ERSGNRTFCWTAV as for subtype 2f (SEQ ID NO 160)
ELQGNKSRCWIPV as for subtype 2g (SEQ ID NO 162)
ERHQNQSRCWIPV as for subtype 2h (SEQ ID NO 163)
EWKDNTSRCWIPV as for subtype 2i (SEQ ID NO 164)
EREGNSSRCWIPV as for subtype 2k (SEQ ID NO 165)
VREGNQSRCWVAL or VRTGNQSRCWVAL or VRVGNQSSCWVAL or
VRVGNQSRCWVAL or VKEGNHSRCWVAL as for subtype 4k (SEQ ID NO 166, 167, 168
or 169)
VKTGNTSRCWVAL as for subtype 41 (SEQ ID NO 170)
IKAGNESRCWLPV as for type 9 (SEQ ID NO 171)
VKEGNQSRCWVQA as for subtype 7c (SEQ ID NO 172)
VKXXNLTKCWLSA as for subtype 7d (SEQ ID NO 173)
VRSGNTSRCWIPV as for type 10 (SEQ ID NO 174)
VKNASVPTAA or VKDANVPTAA as for subtype 1d (SEQ ID NO 175 and 176)
ARIANAPIDE as for subtype 1f (SEQ ID NO 177)
VSKPGALTKG as for subtype 2e (SEQ ID NO 178)
VSRPGALTRG as for subtype 2f (SEQ ID NO 179)
VNQPGALTRG as for subtype 2g (SEQ ID NO 180)
VSQPGALTRG as for subtype 2h (SEQ ID NO 181)
VSQPGALTKG as for subtype 2i (SEQ ID NO 182)
VSRPGALTEG as for subtype 2k (SEQ ID NO 183)
APYIGAPLES or APYTAAPLES as for subtype 4k (SEQ ID NO 184 and 185)
APILSAPLMS as for subtype 4l (SEQ ID NO 186)
VPNSSVPIHG as for type 9 (SEQ ID NO 187)
VPNASTPVTG as for subtype 7c (SEQ ID NO 188)
VQNASVSIRG as for subtype 7d (SEQ ID NO 189)
VKSPCAATAS as for type 10 (SEQ ID NO 190)
SPRMHHTTQE or SPRLYHTTQE as for subtype 1d (SEQ ID NO 191 and 192)
TSRRHWTVQD as for subtype 1f (SEQ ID NO 193)
APKRHYFVQE as for subtype 2e (SEQ ID NO 194)
SPQYHTFVQE as for subtype 2f (SEQ ID NO 195)
SPQHHNFSQD as for subtype 2g (SEQ ID NO 196)
SPQHHIFVQD as for subtype 2h (SEQ ID NO 197)
SPEHHHFVQD as for subtype 2k (SEQ ID NO 198)
RPRRHWTTQD or RPRRHWTAQD or QPRRHWTTQD or RPRRHWTTQE as for subtype 4k (SEQ
ID NO 199, 200, 201 or 202)
QPRRHWTVQD as for subtype 4l (SEQ ID NO 203)
RPKYHQVTQD as for type 9 (SEQ ID NO 204)
RPRMHQVVQE as for subtype 7c (SEQ ID NO 205)
RPRMYEIAQD as for subtype 7d (SEQ ID NO 206)
RHRQHWTVQD as for type 10 (SEQ ID NO 207)
or a part of said polynucleic acid which is unique to at least one of the
HCV subtypes or types as defined Table 5, and which contains at least one
nucleotide differing from known HCV nucleotide sequences, or the
complement thereof.
Using the 5' non-coding LiPA system (Stuyver et al., 1993) and a new core
LiPA system including multiple probes for subtypes 1a, 1b, 1c, 2a, 2b or
2c derived from the core region (Stuyver et al., 1995), samples from the
Benelux, Cameroon, France and Vietnam were selected because of their
aberrant reactivities (isolates CAM 1078, FR2, FR1, VN4, VN12, VN13,
NE98). Some samples were, together with many other samples, sequenced as a
control for typing. Sequencing results, however, indicated the discovery
of new subtypes (isolates BNL1, BNL2, BNL3, FR4, BNL4, BNL5, BNL6, BNL7,
BNL8, BNL9, BNL10, BNL11 and BNL12). Nucleotide sequences in the core and
E1 regions which have not yet been reported before, were analyzed in the
frame of the invention. Genomic sequences of subtype 1d, 1e, 1f, 1g 2e,
2f, 2g, 2h, 2i, 2k, 2l, 3g, 4k, 4l, 4m, 7a, 7c, 7d and types 9, 10 and 11
isolates are reported for the first time in the present invention. The
NS5B region was also analyzed.
The term "polynucleic acid" refers to a single- stranded or double-stranded
nucleic acid sequence which may contain at least 5 contiguous nucleotides
in common with the complete nucleotide sequence (e.g. at least 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35,
40, 45, 50, 60, 75 or more contiguous nucleotides). A polynucleic acid
which is up till about 100 nucleotides in length is often also referred to
as an oligonucleotide. A polynucleic acid may consist of
deoxyribonucleotides or ribonucleotides, nucleotide analogues or modified
nucleotides, or may have been adapted for therapeutic purposes. A
polynucleic acid may also comprise a double stranded cDNA clone which can
be used for cloning purposes, or for in vivo therapy, or prophylaxis.
The oligonucleotides according to the present invention, used as primers or
probes may also contain or consist of nucleotide analogous such as
phosphorothioates (Matsukura et al., 1987), alkylphosphoriates (Miller et
al., 1979) or peptide nucleic acids (Nielsen et al., 1991; Nielsen et al.,
1993) or may contain interculating agents (Asseline et al., 1984).
As most other variations or modifications introduced into the original DNA
sequences of the invention these variations will neccissitate adaptions
with respect to the conditions under which the oligonucleotide should be
used to obtain the required specificty and sensitivity. However the
eventual results will be essentially the same as those obtained with the
unmodified oligonucleotides.
The introduction of these modifications may be advantageous in order to
positivily influence characteristics such as hybridization kinetics,
reversibility of the hybrid-formation, biological stability of the
oligonucleotide molecules, etc.
The polynucleic acids of the invention may be comprised in a composition of
any kind. Said composition may be for diagnostic, therapeutic or
prophylactic use.
The expression "sequences which are unique to an HCV type or subtype"
refers to sequences which are not shared by any other type or subtype of
HCV, and can thus be used to uniquely detect that HCV type or subtype.
Sequence variability is demonstrated in the present invention between the
newly found HCV types and subtypes (see Table 5) and the known HCV types
and subtypes (see Table 3), and it is therefore from these regions of
sequence variability in particular that type- or subtypes-specific
polynucleic acids, oligonucleotides, polypeptides and peptides may be
obtained. The term type- or subtypes-specific refers to the fact that a
sequence is unique to that HCV type or subtype involved.
The expression "nucleotides corresponding to" refers to nucleotides which
are homologous or complementary to an indicated nucleotide sequence or
region within a specific HCV sequence.
The term "coding region" corresponds to the region of the HCV genome that
encodes the HCV polyprotein. In fact, it comprises the complete genome
with the exception of the 5' untranslated region and 3' untranslated
region.
The term "HCV polyprotein" refers to the HCV polyprotein of the HCV-J
isolate (Kato et al., 1990). The adenine residue at position 330 (Kato et
al., 1990) is the first residue of the ATG codon that initiates the long
HCV polyprotein of 3010 amino acids in HCV-J and other type 1b isolates,
and of 3011 amino acids in HCV-1 and other type 1a isolates, and of 3033
amino acids in type 2 isolates HC-J6 and HC-J8 (Okamoto et al., 1992).
This adenine is designated as position 1 at the nucleic acid level, and
this methionine is designated as position 1 at the amino acid level, in
the present invention. As type 1a isolates contain 1 extra amino acid in
the NS5A region, coding sequences of type 1a and 1b have identical
numbering in the Core, E1, NS3, and NS4 region, but will differ in the
NS5B region as indicated in Table 1. Type 2 isolates have 4 extra amino
acids in the E2 region, and 17 or 18 extra amino acids in the NS5 region
compared to type 1 isolates, and will differ in numbering from type 1
isolates in the NS3/4 region and NS5b regions as indicated in Table 1.
Similar insertions compared with type 1 (but of a different size) can also
be observed in type 3a sequences which affect the numbering of type 3a
amino acids accordingly. Other insertions or deletions may be readily
observed in type 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11 sequences after
alignment withknown HCV sequences.
TABLE 1
Positions Positions Positions Positions
described described for described for described for
in the HCV-J HCV-1 HC-J6, HC-
present (Kato et al., (Choo et al., J8 (Okamoto
Region invention* 1990) 1991) et al., 1992)
Nucleo- NS5B 8023/8235 8352/8564 8026/8238 8433/8645
tides 7932/8271 8261/8600 7935/8274 8342/8681
coding 330/9359 1/9033 342/9439
region
of present
invention
Amino NS5B 2675/2745 2675/2745 2676/2746 2698/2768
Acids 2645/2757 2645/2757 2646/2758 2668/2780
Table 1: Comparison ot the HCV nucleotide and amino acid numbering system
used in the present invention (*) with the numbering used for other
prototype isolates. For example, 8352/8564. indicates the region
designated by the numbering from nucleotide 8352 to nucleotide 8564 as
described by Kato et al. (1990). Since the numbering system of the present
invention starts at the polyprotein initiation site, the 329
# nucleotides of the 5' untranslated region described by Kato et al. (1990)
have to be substracted, and the corresponding region is numbered from
nucleotide 8023 (`8352-329`) to 8235 (`8564-329`).
The term "genotype" as used in the present invention refers to both types
and/or subtypes.
The term "HCV type" corresponds to a group of HCV isolates of which the
complete genome shows more than 73% preferably more than 74% homology at
the nucleic acid level, or of which the NS5 region between nucleotide
positions 7932 and 8271 shows more than 75.4% homology at the nucleic acid
level, or of which the complete HCV polyprotein shows more than 78%
homology at the amino acid level, or of which the NS5 region between amino
acids at positions 2645 and 2757 shows more than 80% homology at the amino
acid level, to polyproteins of the other isolates of the group, with said
numbering beginning at the first ATG codon or first methionine of the long
HCV polyprotein of the HCV-J isolate (Kato et al., 1990). Isolates
belonging to different types of HCV exhibit homologies, over the complete
genome, of less than 74%, preferably less than 73%, at the nucleic acid
level and less than 78% at the amino acid level. Isolates belonging to the
same type usually show homologies of about 90 to 99% at the nucleic acid
level and 95 to 96% at the amino acid level when belonging to the same
subtype, and those belonging to the same type but different subtypes
preferably show homologies of about 76% to 82% (more particularly of about
77% to 80%) at the nucleic acid level and 85-86% at the amino acid level.
More preferably the definition of HCV types is concluded from the
classification of HCV isolates according to their nucleotide distances
calculated as detailed below:
(1) based on phylogenetic analysis of nucleic acid sequences in the NS5B
region between nucleotides 7935 and 8274 (Choo et al., 1991) or 8261 and
8600 (Kato et al., 1990) or 8342 and 8681 (Okamoto et al., 1991), isolates
belonging to the same HCV type show nucleotide distances of less than
0.34, usually less than 0.33, and more usually of less than 0.32, and
isolates belonging to the same subtype show nucleotide distances of less
than 0.135, usually of less than 0.13, and more usually of less than
0.125, usually ranging between 0.0003 and 0.1151, and consequently
isolates belonging to the same type but different subtypes show nucleotide
distances ranging from 0.135 to 0.34, usually ranging from 0.1384 to
0.2977, and more usually ranging from 0.15 to 0.32, and isolates belonging
to different HCV types show nucleotide distances greater than 0.34,
usually greater that 0.35, and more usually of greater than 0.358, more
usually ranging from 0.3581 to 0.6670.
(2) based on phylogenetic analysis of nucleic acid sequences in the core/E1
region between nucleotides 378 and 957, isolates belonging to the same HCV
type show nucleotide distances of less than 0.38, usually of less than
0.37, and more usually of less than 0.364, and isolates belonging to the
same subtype show nucleotide distances of less than 0.1 7, usually of less
than 0.16, and more usually of less than 0.15, more usually less than
0.135, more usually less than 0.134, and consequently isolates belonging
to the same type but different subtypes show nucleotide distances ranging
from 0.15 to 0.38, usually ranging from 0.16 to 0.37, and more usually
ranging from 0.17 to 0.36, more usually ranging from 0.133 to 0.379, and
isolates belonging to different HCV types show nucleotide distances
greater than 0.34, 0.35, 0.36, usually more than 0.365, and more usually
of greater than 0.37,
TABLE 2
Molecular evolutionary distances
Core/E1 E1 NS5B NS5B
Region 579 bp 384 bp 340 bp 222 bp
Isolates* 0.0017-0.1347 0.0026-0.2031 0.0003-0.1151 0.000-0.1323
(0.0750 .+-. 0.0245) (0.0969 .+-. 0.0289) (0.0637 .+-. 0.0229)
(0.0607 .+-. 0.0205)
Subtypes* 0.1330-0.3794 0.1645-0.4869 0.1384-0.2977 0.117-0.3538
(0.2786 .+-. 0.0363) (0.3761 .+-. 0.0433) (0.2219 .+-. 0.0341)
(0.2391 .+-. 0.0399)
Types* 0.3479-0.6306 0.4309-0.9561 0.3581-0.6670 0.3457-0.7471
(0.4703 .+-. 0.0525) (0.6309 .+-. 0.0928) (0.4994 .+-. 0.0495)
(0.5295 .+-. 0.0627)
Table 2 *Figures created by the PHYLIP program DNADIST are expressed as
minimum to maximum (average .+-. standard deviation). Phylogenetic
distances for isolates belonging to the same subtype (`isolates`), to
different subtypes of the same type (`subtypes`), and to different types
(`types`) are given.
In a comparative phylogenetic analysis of available sequences, ranges of
molecular evolutionary distances for different regions of the genome were
calculated, based on 19,781 pairwise comparisons by means of the DNADIST
program of the phylogeny inference package PHYLIP version 3.5c
(Felsenstein, 1993). The results are shown in Table 2 and indicate that
although the majority of distances obtained in each region fit with
classification of a certain isolate, only the ranges obtained in the 340bp
NS5B-region are non-overlapping and therefore conclusive. However, as was
performed in the present invention, it is preferable to obtain sequence
information from at least 2 regions before final classification of a given
isolate.
Designation of a number to the different types of HCV and HCV nomenclature
is based on chronological discovery of the different types. The numbering
system used in the present invention might still fluctuate according to
international conventions or guidelines. For example, "type 4" might be
changed into "type 5" or "type 6". Also the arbitrarily chosen border
distances between types and subtypes and isolates may still be subject to
change according to international guidelines or conventions. Therefore
types 7a, 8a, 8b, 9a may for example be designated 6b, 6c, 6d, and 6d in
the future; and type 10a which shows relatedness with genotype 3 may be
denoted 3g instead of 10a.
The term "subtype" corresponds to a group of HCV isolates of which the
complete polyprotein shows a homology of more than 90% both at the nucleic
acid and amino acid levels, or of which the NS5 region between nucleotide
positions 7932 and 8271 shows a homology of more than 90% at the nucleic
acid level to the corresponding parts of the genomes of the other isolates
of the same group, with said numbering beginning with the adenine residue
of the initiation codon of the HCV polyprotein. Isolates belonging to the
same type but different subtypes of HCV show homologies of more than 74%
at the nucleic acid level and of more than 78% at the amino acid level.
It is to be understood that extremely variable regions such as the E1, E2
and NS4 regions will exhibit lower homologies than the average homology of
the complete genome of the polyprotein.
Using these criteria, HCV isolates can be classified into at least 11
types.
Several subtypes can clearly be distinguished in types 1, 2, 3, 4 and 7:
1a, 1b, 1c, 1d, 1e, 1f, 1g, 2a, 2b, 2c, 2d, 2e, 2f, 2g, 2h, 2i, 2k, 2l,
3a, 3c, 3d, 3f, 3g, 4a, 4b, 4c, 4d, 4e, 4f, 4g, 4h, 4i, 4j, 4k, 4l, 4m,
7a, 7c, and 7c, and 7d based on homologies of the 5' UR and coding
regions. An overview of most of the reported isolates and their proposed
classification according to the typing system of the present invention as
well as other proposed classifications is presented in Table 3.
TABLE 3
HCV CLASSIFICATION
OKA- NA-
MOTO MORI CHA KAO PROTOTYPE
1a I I Pt GI HCV-1, HCV-H, HC-J1
1b II II KI GII HCV-J, HCV-BK, HCV-T,
HC-JK1, HC-J4, HCV-CHINA
1c HC-G9
2a III III K2a GIII HC-36
2b IV IV K2b GIlI HC-J8
2c S83, ARG6, ARG8, I10, T983
2d NE92
3a V V K3 GIV BR36, BR56, HD10, N2L1, BR33,
Ta, E-b1
3b VI K3 GIV HCV-TR, Tb, NE137
3c NE48
3d NE274
3e NE145
3f NE125
4a Z4, GB809-4
4b Z1
4c GB116, GB358, GB215, Z6, Z7
4d DK13
4e GB809-2, CAM600, CAM736
4f CAM622, CAM627
4g GB549
4h GB438
4i CAR4/1205
4j CAR1/905
5a GV SA3, SA4, SA1, SA7, SA11,
BE95 HK1, HK2, HK3, HK4,
VN11
Table 3 Overview of the known HCV types and subtypes classified according
to the different authors.
The term "complement" refers to a nucleotide sequence which is
complementary to an indicated sequence and which is able to hybridize to
the indicated sequences.
The composition of the invention can comprise many combinations. By way of
example, the composition of the invention can comprise:
two (or more) nucleic acids from the same region or,
two nucleic acids (or more), respectively from different regions, for the
same isolate or for different isolates,
or nucleic acids from the same regions and from at least two different
regions (for the same isolate or for different isolates).
The present invention relates particularly to a polynucleic acid as defined
above having a sequence selected from any of SEQ ID NO 1, 3, 5, 7, 9, 11,
13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39 , 41, 43, 45, 47,
49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83,
85, 87, 89, 91, 93, 95, 97, 99, 101, 103 to 105, or a part of said
polynucleic acid which is unique to any of the HCV subtypes or types as
defined in Table 5, and which contains at least one nucleotide differing
from known HCV polynucleic acids, or the complement thereof.
The present invention relates more particularly to a polynucleic acid as
defined above, which codes for the 5' UR, the Core/El, the NS4 or the NS5B
region or a part thereof.
More particularly, the present invention relates to a polynucleic acid as
defined above which is a cDNA sequence.
Also included within the present invention are sequence variants of the
polynucleic acids as selected from any of the nucleotide sequences as
given in any of the above given SEQ ID numbers with said sequence variants
containing either deletion and/or insertions of one or more nucleotides,
especially insertions or deletions of 1 or more codons, mainly at the
extremities of oligonucleotides (either 3' or 5'), or substitutions of
some non-essential nucleotides (i.e. nucleotides not essential to
discriminate between different genotypes of HCV) by others (including
modified nucleotides an/or inosine), for example, a type 1 or 2 sequence
might be modified into a type 7 sequence by replacing some nucleotides of
the type 1 or 2 sequence with type-specific nucleotides of type 7 as shown
in for instance FIG. 1 and 2.
Particularly preferred variant polynucleic acids of the present invention
include also sequences which hybridise under stringent conditions with any
of the polynucleic acid sequences of the present invention. Particularly,
sequences which show a high degree of homology (similarity) to any of the
polynucleic acids of the invention as described above. Particularly
sequences which are at least 80%, 85%, 90%, 95% or more homologous to said
polynucleic acid sequences of the invention. Preferably said sequences
will have less than 20%, 15%, 10%, or 5% variation of the original
nucleotides of said polynucleic acid sequence.
Polynucleic acid sequences according to the present invention which are
homologous to the sequences as represented by a SEQ ID NO can be
characterized and isolated according to any of the techniques known in the
art, such as amplification by means of sequence-specific primers,
hybridization with sequence-specific probes under more or less stringent
conditions, serological screening methods or via the LiPA typing system.
Other preferred variant polynucleic acids of the present invention include
sequences which are redundant as a result of the degeneracy of the genetic
code compared any of the above-given polynucleic acids of the present
invention. These variant polynucleic acid sequences will thus encode the
same amino acid sequence as the polynucleic acids they are derived from.
Also included within the scope of the present invention are 5' non-coding
region sequences which can be readily obtained from type 1 subtype 1d, 1e,
1f or 1g isolates; type 2 subtype 2e, 2f, 2g, 2h, 2i, 2k or 2l isolates;
type 3 subtype 3g isolates; type 4 subtype 4k, 4l or 4m isolates; type 7
subtype 7a, 7c or 7d isolates, type 9, type 10 or type 11 isolates
discribed herein. Such sequences may contain type or subtype-specific
motifs which can be employed for type and/or subtype-specific
hybridization assays, e.g. such as described by Stuyver et al. (1993).
Polynucleic acid sequences of the genomes indicated above from regions not
yet depicted in the present examples, figures and sequence listing can be
obtained by any of the techniques known in the art, such as amplification
techniques using suitable primers from the sequences of these new genomes
given in FIG. 1 of the present invention.
The present invention also relates to an oligonucleotide primer comprising
part of a polynucleic acid as defined above, with said primer being able
to act as a primer for specifically amplifying the nucleic acid of a
certian HCV isolate belonging to the genotype from which the primer is
derived.
The term "primer" refers to a single stranded DNA oligonucleotide sequence
capable of acting as a point of initiation for synthesis of a primer
extension product which is complementary to the nucleic acid strand to be
copied. The length and the sequence of the primer must be such that they
allow to prime the synthesis of the extension products. Preferably the
primer is about 5-50 nucleotides. Specific length and sequence will depend
on the complexity of the required DNA or RNA targets, as well as on the
conditions of primer such as temperature and ionic strength.
The fact that amplification primers do not have to match exactly with
corresponding template sequence to warrant proper amplification is amply
documented in the literature (Kwok et al., 1990).
The amplification method used can be either polymerase chain reaction (PCR;
Saiki et al., 1988), ligase chain reaction (LCR; Landgren et al., 1988; Wu
& Wallace, 1989; Barany, 1991), nucleic acid sequence-based amplification
(NASBA; Guatelli et al., 1990; Compton, 1991), transcription-based
amplification system (TAS; Kwoh et al., 1989), strand displacement
amplification (SDA; Duck, 1990; Walker et al., 1992) or amplification by
means of Q.beta. replicase (Lizardi et al., 1988; Lomeli et al., 1989) or
any other suitable method to amplify nucleic acid molecules using primer
extension. During amplification, the amplified products can be
conveniently labelled either using labelled primers or by incorporating
labelled nucleotides. Labels may be isotopic (.sup.32 P, .sup.35 S, etc.)
or non-isotopic (biotin, digoxigenin, etc.). The amplification reaction is
repeated between 20 and 70 times, advantageously between 25 and 45 times.
The present invention also relates to an oligonucleotide probe comprising
part of a polynucleic acid as defined above, with said probe being able to
act as a hybridization probe for specific detection and/or classification
into types and/or subtypes of an HCV nucleic caid containing said
nucleotide sequence, with said probe being possibly labelled or attached
to a solid substrate.
The term "probe" refers to single stranded sequence-specific
oligonucleotides which have a sequence which is complementary to the
target sequence of the HCV genotype(s) to be detected.
Preferably, these probes are about 5 to 50 nucleotides long, more
preferably from about 10 to 25 nucleotides.
The term "solid support" can refer to any substrate to which an
oligonucleotide probe can be coupled, provided that it retains its
hybridization characteristics and provided that the background level of
hybridization remains low. Usually the solid substrate will be a
microtiter plate, a membrane (e.g. nylon or nitrocellulose) or a
microsphere (bead). Prior to application to the membrane or fixation it
may be convenient to modify the nucleic acid probe in order to facilitate
fixation or improve the hybridization efficiency. Such modifications may
encompass homopolymer tailing, coupling with different reactive groups
such as aliphatic groups, NH.sub.2 groups, SH groups, carboxylic groups,
or coupling with biotin or haptens.
The present invention also relates to a diagnostic kit for use in
determining the genotype of HCV, said kit comprising a primer as defined
above.
The present invention also relates to a diagnostic kit for use in
determining the genotype of HCV, said kit comprising a probe as defined
above.
The present invention also relates to a diagnostic kit as defined above,
wherein said probe(s) is(are) attached to a solid substrate.
The present invention also relates to a diagnostic kit as defined above,
wherein a range of said probes is attached to specific locations on a
solid substrate.
The present invention also relates to a diagnostic kit as defined above,
wherein said solid support is a membrane strip and said probes are coupled
to the membrane in the form of parallel lines.
The present invention also relates to a method for the detection of HCV
nucleic acids present in a biological sample, comprising:
(i) possibly extracting sample nucleic acid,
(ii) amplifying the nucleic acid with at least one primer as defined above,
(iii) detecting the amplified nucleic acids.
The present invention also relates to a method for the detection of HCV
nucleic acids present in a biological sample, comprising:
(i) possibly extracting sample nucleic acid,
(ii) possibly amplifying the nucleic acid with at least one primer as
defiend above, or with a universal HCV primer,
(iii) hybridizing the nucleic acids of the biological sample, possibly
under denatured conditions, at appropriate conditions with one or more
probes as defined above, with said probes being preferably attached to a
solid substrate,
(iv) possibly washing at appropriate conditions,
(v) detecting the hybrids formed.
The present invention also relates to a method for detecting the presence
of one or more HCV genotypes present in a biological sample, comprising:
(i) possibly extracting sample nucleic acid,
(ii) specifically amplifying the nucleic acid with at least one primer as
defined above,
(iii) detecting said amplified nucleic acids.
The present invention also relates to a method for detecting the presence
of one or more HCV genotypes present in a biological sample, comprising:
(i) possibly extracting sample nucleic acid,
(ii) possibly amplifying the nucleic acid with at least one primer as
defined above or with a universal HCV primer,
(iii) hybridizing the nucleic acids of the biological sample, possibly
under denatured conditions, at appropriate conditions with one or more
probes as defined above, with said probes being preferably attached to a
solid substrate,
(iv) possibly washing at appropriate conditions,
(v) detecting the hybrids formed,
(vi) inferring the presence of one or more HCV genotypes present from the
observed hybridization pattern.
The present invention also relates to a method as defined above, wherein
said probes are further characterized as defined above.
The present invention also relates to a method as defined above, wherein
said nucleic acids are labelled during or after amplification.
Preferably, this technique could be performed in the 5' non-coding, Core or
NS5B region.
The term "nucleic acid" can also be referred to as analyte strand and
corresponds to a single- or double-stranded nucleic acid molecule. This
analyte strand is preferentially positive- or negative stranded RNA, cDNA
or amplified cDNA.
The term "biological sample" refers to any biological sample (tissue or
fluid) containing HCV nucleic acid sequences and refers more particularly
to blood serum or plasma samples.
The term "universal HCV primer" refers to oligonucleotide sequences
complementary to any of the conserved regions of the HCV genome.
The expression "appropriate" hybridization and washing conditions are to be
understood as stringent and are generally known in the art (e.g. Maniatis
et al., Molecular Cloning: A Laboratory Manual, New York, Cold Spring
Harbor Laboratory, 1982).
However, according to the hybridization solution (SSC, SSPE, etc.), these
probes should be hybridized at their appropriate temperature in order to
attain sufficient specificity.
The term "labelled" refers to the use of labelled nucleic acids. This may
include the use of labelled nucleotides incorporated during the polymerase
step of the amplification such as illustrated by Saiki et al. (1988) or
Bej et al. (1990) or labelled primers, or by any other method known to the
person skilled in the art.
The process of the invention comprises the steps of contacting any of the
probes as defined above, with one of the following elements:
either a biological sample in which the nucleic acids are made available
for hybridization,
or the purified nucleic acids contained in the biological sample
or a single copy derived from the purified nucleic acids,
or an amplified copy derived from the purified nucleic acids, with said
elements or with said probes being attached to a solid substrate.
The expression "inferring the presence of one or more HCV genotypes present
from the observed hybridization pattern" refers to the identification of
the presence of HCV genomes in the sample by analyzing the pattern of
binding of a panel of oligonucleotide probes. Single probes may provide
useful information concerning the presence or absence of HCV genomes in a
sample. On the other hand, the variation of the HCV genomes is dispersed
in nature, so rarely is any one probe able to identify uniquely a specific
HCV genome. Rather, the identity of an HCV genotype may be inferred from
the pattern of binding of a panel of oligonucleotide probes, which are
specific for (different) segments of the different HCV genomes. Depending
on the choice of these oligonucleotide probes, each known HCV genotype
will correspond to a specific hybridization pattern upon use of a specific
combination of probes. Each HCV genotype will also be able to be
discriminated from any other HCV genotype amplified with the same primers
depending on the choice of the oligonucleotide probes. Comparison of the
generated pattern of positively hybridizing probes for a sample containing
one or more unkown HCV sequences to a scheme of expected hybridization
patterns, allows one to clearly infer the HCV genotypes present in said
sample.
The present invention thus relates to a method as defined above, wherein
one or more hybridization probes are selected from any of SEQ ID NO 1, 3,
5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,
43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77,
79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103 or 105 or sequence
variants thereof as defined above.
In order to distinguish the amplified HCV genomes from each other, the
target polynucleic acids are hybridized to a set of sequence-specific DNA
probes targetting HCV genotypic regions (unique regions) located in the
HCV polynucleic acids.
Most of these probes target the most type- or subtype-specific regions of
HCV genotypes, but some can be caused to hybridize to more than one HCV
genotype.
According to the hybridization solution (SSC, SSPE, etc.), these probes
should be stringently hybridized at their appropriate temperature in order
to attain sufficient specificity. However, by slightly modifying the DNA
probes, either by adding or deleting one or a few nucleotides at their
extremities (either 3' or 5'), or substituting some non-essential
nucleotides (i.e. nucleotides not essential to discriminate between types)
by others (including modified nucleotides or inosine) these probes or
variants thereof can be caused to hybridize specifically at the same
hybridization conditions (i.e. the same temperature and the same
hybridization solution). Also changing the amount (concentration) of probe
used may be beneficial to obtain more specific hybridization results. It
should be noted in this context, that probes of the same length,
regardless of their GC content, will hybridize specifically at
approximately the same temperature in TMACI solutions (Jacobs et al.,
1988).
Suitable assay methods for purposes of the present invention to detect
hybrids formed between the oligonucleotide probes and the nucleic acid
sequences in a sample may comprise any of the assay formats known in the
art, such as the conventional dot-blot format, sandwich hybridization or
reverse hybridization. For example, the detection can be accomplished
using a dot blot format, the unlabelled amplified sample being bound to a
membrane, the membrane being incorporated with at least one labelled probe
under suitable hybridization and wash conditions, and the presence of
bound probe being monitored.
An alternative and preferred method is a "reverse" dot-blot format, in
which the amplified sequence contains a label. In this format, the
unlabelled oligonucleotide probes are bound to a solid support and exposed
to the labelled sample under appropriate stringent hybridization and
subsequent washing conditions. It is to be understood that also any other
assay method which relies on the formation of a hybrid between the nucleic
acids of the sample and the oligonucleotide probes according to the
present invention may be used.
According to an advantageous embodiment, the process of detecting one or
more HCV genotypes contained in a biological sample comprises the steps of
contacting amplified HCV nucleic acid copies derived from the biological
sample, with oligonucleotide probes which have been immobilized as
parallel lines on a solid support.
According to this advantageous method, the probes are immobilized in a Line
Probe Assay (LiPA) format. This is a reverse hybridization format (Saiki
et al., 1989) using membrane strips onto which several oligonucleotide
probes (including negative or positive control oligonucleotides) can be
conveniently applied as parallel lines.
The invention thus also relates to a solid support, preferably a membrane
strip, carrying on its surface, one or more probes as defined above,
coupled to the support in the form of parallel lines.
The LiPA is a very rapid and user-friendly hybridization test. Results can
be read after 4 hours. after the start of the amplification. After
amplification during which usually a non-isotopic label is incorporated in
the amplified product, and alkaline denaturation, the amplified product is
contacted with the probes on the membrane and the hybridization is carried
out for about 1 to 1,5 h hybridized polynucleic acid is detected. From the
hybridization pattern generated, the HCV type can be deduced either
visually, but preferably using dedicated software. The LiPA format is
completely compatible with commercially available scanning devices, thus
rendering automatic interpretation of the results very reliable. All those
advantages make the LiPA format liable for the use of HCV detection in a
routine setting. The LiPA format should be particularly advantageous for
detecting the presence of different HCV genotypes.
The present invention also relates to a method for detecting and
identifying novel HCV genotypes, different from the known HCV genomes,
comprising the steps of:
determining to which HCV genotype the nucleotides present in a biological
sample belong, according to the process as defined above,
in the case of observing a sample which does not generate a hybridization
pattern compatible with those defined in Table 3, sequencing the portion
of the HCV genome sequence corresponding to the aberrantly hybridizing
probe of the new HCV genotype to be determined.
The present invention also relates to a method for preparing a polynucleic
acid according to the present invention. These methods include any method
known in the art for preparing polynucleic acids (e.g. the phosphodiester
method for synthesizing oligonucleotides as described by Agarwal et al.
1972, Agnew. Chem. Int. Ed. Engl. 11:451, the phosphotriester method of
Hsiung et al. 1979, Nucleic Acid Res. 6:1371, or the automated
diethylphosphoramidite method of Baeucage et al. 1981, Tetrahedron Letters
22:1859-1862.). Alternatively, the polynucleic acids of the present
invention may be isolated fragments of naturally occuring or cloned DNA or
RNA. In addition, the oligonucleotides according to the present invention
may be synthesized automatically on commercial instruments sold by a
variety of manufacturers.
The present invention particularly also relates to a polypeptide having an
amino acid sequence encoded by a polynucleic acid as defined above, or a
part thereof which is unique to at least one of the HCV subtypes or types
as defined in Table 5, and which contains at least one amino acid
differing from any of the known HCV types or subtypes, or an analog
thereof being substantially homologous and biologically equivalent.
The term `polypeptide` refers to a polymer of amino acids and does not
refer to a specific length of the product; thus, peptides, oligopeptides,
and proteins are included within the definition of polypeptide. This term
also does not refer to or exclude post-expression modifications of the
polypeptide, for example, glycosylations, acetylations, phosphorylations
and the like. Included within the definition are, for example,
polypeptides containing one or more analogues of an amino acid (including,
for example, unnatural amino acids, PNA, etc.), polypeptides with
substituted linkages, as well as other modifications known in the art,
both naturally occurring and non-naturally occurring.
The term "unique" is referred above.
By "biologically equivalent" as used throughout the specification and
claims, it is meant that the compositions are immunogenically equivalent
to the proteins (polypeptides) or peptides of the invention as defined
above and below.
By "substantially homologous" as used throughout the ensuing specification
and claims to describe proteins and peptides, it is meant a degree of
homology in the amino acid sequence to the proteins or peptides of the
invention. Preferably the degree of homology is in excess of 90,
preferably in excess of 95, with a particularly preferred group of
proteins being in excess of 99 homologous with the proteins or peptides of
the invention.
The term "analog" as used throughout the specification or claims to
describe the proteins or peptides of the present invention, includes any
protein or peptide having an amino acid residue sequence substantially
identical to a sequence specifically shown herein in which one or more
residues have been conservatively substituted with a biologically
equivalent residue. Examples of conservative substitutions include the
substitution of one-polar (hydrophobic) residue such as isoleucine,
valine, leucine or methionine for another, the substitution of one polar
(hydrophillic) residue for another such as between arginine and lysine,
between glutamine and asparagine, between glycine and serine, the
substitution of one basic residue such as lysine, arginine or histidine
for another, or the substitution of one acidic residue, such as aspartic
acid or glutamic acid for another. Examples of allowable mutations
acccording to the present invention can be found in Table 4.
The phrase "conservative substitution" also includes the use of a
chemically derivatized residue in place of a non-derivatized residue
provided that the resulting protein or peptide is biologically equivalent
to theprotein or peptide of the invention.
"Chemical derivative" refers to a protein or peptide having one or more
residues chemically derivatized by reaction of a functional side group.
Examples of such derivatized molecules, include but are not limited to,
those molecules in which free amino groups have been derivatized to form
amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups,
t-butyloxycarbonyl groups, chloracetyl groups or formyl groups. Free
carboxyl groups may be derivatized to form salts, methyl and ethyl esters
or other types of esters or hydrazides. Free hydroxyl groups may be
derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen
of histidine may be derivatized to form N-imbenzylhistidine. Also included
as chemical derivatives are those proteins or peptides which contain one
or more naturally-occurring amino acid derivatives of the twenty standard
amino acids. For examples : 4-hydroxyproline may be substituted for
proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine
may be substituted for histidine; homoserine may be substituted for
serine; and ornithine may be substituted for lysine. The proteins or
peptides of the present invention also include any protein or peptide
having one or more additions and/or deletions or residues relative to the
sequence of a peptide whose sequence is shown herein, so long as the
peptide is biologically equivalent to the proteins or peptides of the
invention.
It is to be noted that, at the level of the amino acid sequence, at least
one amino acids difference (with respect to known HCV amino acid
sequences) is sufficient to be part of the invention, which means that the
polypeptides of the invention correspond to polynucleic acids having at
least one nucleotide difference (with known HCV polynucleic acid
sequences) involving an amino acid difference in the encoded polyprotein.
As the NS4 and the Core regions are known to contain several epitopes, for
example characterized in patent application EP-A-0 489 968, and as the E1
protein is expected to be subject to immune attack as part of the viral
envelope and expected to contain epitopes, the NS4, Core and E1 epitopes
of the new types and subtypes disclosed herein will consistently differ
from the epitopes present in previously known genotypes. This is
examplified by the type-specificity of NS4 synthetic peptides as described
in Simmonds et al. (1993c) and Stuyver et al. (1993b) and PCT/EP 94/01323
and the type-specificity of recombinant E1 proteins as described in
Maertens et al. (1994).
The peptides according to the present invention contain preferably at least
3, preferably 4, 5 contiguous HCV amino acids, 6, 7 preferably however at
least 8 contiguous HCV amino acids, at least 10 or at least 15 (for
instance at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23,
24, 25, 30, 35, 40, 45, 50 or more amino acids).
TABLE 4
Amino acids Synonymous groups
Ser (S) Ser, Thr, Gly, Asn
Arg (R) Arg, His, Lys, Glu, Gln
Leu (L) Leu, Ile, Met, Phe, Val, Tyr
Pro (P) Pro, Ala, Thr, Gly
Thr (T) Thr, Pro, Ser, Ala, Gly; His, Gln
Ala (A) Ala, Pro, Gly, Thr
Val (V) Val, Met, Ile, Tyr, Phe, Leu, Val
Gly (G) Gly, Ala, Thr, Pro, Ser
Ile (i) Ile, Met, Leu, Phe, Val, Ile, Tyr
Phe (F) Phe, Met, Tyr, Ile, Leu, Trp, Val
Tyr (Y) Tyr, Phe, Trp, Met, Ile, Val, Leu
Cys (C) Cys, Ser, Thr, Met
His (H) His, Gln, Arg, Lys, Glu, Thr
Gln (Q) Gin, Glu, His, Lys, Asn, Thr, Arg
Asn (N) Asn, Asp, Ser, Gln
Lys (K) Lys, Arg, Glu, Gln, His
Asp (D) Asp, Asn, Glu, Gln
Glu (E) Glu, Gln, Asp, Lys, Asn, His, Arg
Met (M) Met, Ile, Leu, Phe, Val
Table 4 Overview of the amino acid substitutions which could form the basis
of analogs (muteins) as defined above
The polypeptides of the invention, and particularly the fragments, can be
prepared by classical chemical synthesis.
The synthesis can be carried out in homogeneous solution or in solid phase.
For instance, the synthesis technique in homogeneous solution which can be
used is the one described by Houbenweyl in the book entitled "Methode der
organischen chemie" (Method of organic chemistry) edited by E. Wunsh, vol.
15-I et II. THIEME, Stuttgart 1974.
The polypeptides of the invention can also be prepared in solid phase
according to the methods described by Atherton and Shepard in their book
entitled "Solid phase peptide synthesis" (IRL Press, Oxford, 1989).
The polypeptides according to this invention can be prepared by means of
recombinant DNA techniques as described by Maniatis et al., Molecular
Cloning: A Laboratory Manual, New York, Cold Spring Harbor Laboratory,
1982).
The present invention relates particularly to a polypeptide as defined
above, comprising in its amino acid sequence at least one of the following
amino acid residues:
I15, C38, V44, A49, Q43, P49, Q55, A58, S60 or D60, E68 or V68, H70, A71 or
Q71 or N71, D72, H81, H101, D106, S110, L130, I134, E135, L140, S148, T150
or E150, Q153, F155, D157, G160, E165, I169, F181, L186, T190, T192 or
I192 or H192, I193, A195, S196, R197 or N197 or K197, Q199 or D199 or
H199, N199, F200 or T200, A208, I213, M216 or S216, N217 or S217 or G217
or K217, T218, I219, A222, Y223, I230, W231 or L231, S232 or H232 or A232,
Q233, E235 or L235, F236 or T236, F237, L240 or M240, A242, N244, N249,
I250 or K250 or R250, A252 or C252, A254, I255 or V255, D256 or M256,
E257, E260 or K260, R261, V268, S272 or R272, I285, G290 or F290, A291,
A293 or L293 or W293, T294 or A294, S295, H295, K296 or E296, Y297 or
M297, I299 or Y299, I300, S301, P316, S2646, A2648, G2649, A2650, V2652,
Q2653, H2656 or L2656, D2657, F2659, K2663 or Q2663, A2667 or V2667,
D2677, L2681, M2686 or Q2686 or E2686, A2692 or K2692, H2697, I2707, L2708
or Y2708, A2709, A2719 or M2719, F2727, T2728 or D2728, E2729, F2730 or
Y2730, I2741, I2745, V2746 or E2746 or L2746 or K2746, A2748, S2749 or
P2749, R2750, E2751, D2752 or N2752 or S2752 or T2752 or V2752 or I2752 or
Q2752, S2753 or D2753 or G2753, D2754, A2755, L2756 or Q2756, or R2757,
with said notation being composed of a letter representing the amino acid
residue by its one-letter code, and a number representing the amino acid
numbering according to Kato et al., 1990 as shown in Table 1 (see also the
numbering in FIGS. 2, 4 and 6), or a part thereof which is unique to at
least one of the HCV subtypes or types as defined in Table 5, and which
contains at least one amino acid differing from any of the known HCV types
or subtypes, or an analog thereof being substantially homologous and
biologically equivalent to said polypeptide or part thereof.
These unique amino acid residues can be deduced from aligning the new HCV
amino acid sequences as given in FIG. 3 to all known HCV sequences. An
alignment with the new sequences as represented in SEQ ID NO 1 to 106 is
given in for instance FIGS. 2, 4 and 6. It should be clear that the
alignments given in these figures may be completed with all known HCV
sequences to illustrate that any of the above-given unique residues is
indeed unique for at least one of the new HCV sequences of the present
invention.
Within the group of unique and new amino acid residues of the present
invention, unique residues may be found which are specific for the
following new types (subtypes) of HCV according to the HCV classification
system used in the present invention: type 1 subtype 1d, 1e, if or 1g
isolates; type 2 subtype 2e, 2f, 2g, 2h, 2i, 2k or 2l isolates; type 3
subtype 3g isolates; type 4 subtype 4k, 4l or 4m isolates; type 7 subtype
7a, 7c or 7d isolates, type 9, type 10 or type 11 isolates. In order to
obtain these residues the alignments given in FIGS. 2, 4 and 6 may be used
to deduce the type- and or subtype-specificity of any of the unique
residues given above.
For example T190 (detected in subtype 1d) refers to a threomine at position
190 (see FIG. 2). In other sequences only a serine (S190) or exceptionally
an alanine (A190 in type 10a) can be detected.
The polypeptides according to this embodiment of the invention may be
possibly labelled, or attached to a solid substrate, or coupled to a
carrier molecule such as biotin, or mixed with a proper adjuvant all known
in the art and according to the intended use (diagnostic, therapeutic or
prophylactic).
The present invention also relates to a polypeptide as defined above,
comprising in its amino acid sequence at least one of the sequences
repesented by SEQ ID NO107 to 207 as listed above, or a part thereof which
is unique to at least one of the HCV subtypes or types as defined in Table
5, or an analog thereof being substantially homologous and biologically
equivalent to said polypeptide or part thereof.
The present invention relates also to a polypeptide having an amino acid
sequence as represented in any of SEQ ID NO 1 to 106, or a part thereof
which is unique to at least one of the HCV subtypes or types as defined in
Table 5, or an analog thereof being substantially homologous and
biologically equivalent to said polypeptide or part thereof.
The variable region in the core protein (V-CORE in FIG. 2) has been shown
to be useful for serotyping (Machida et al., 1992). The sequence of the
type 1 subtype 1d, 1e, 1f or 1g sequence; type 2 subtype 2e, 2f, 2g, 2h,
2i, 2k and 2l sequence; type 3 subtype 3g; type 4, subtype 4k, 4l or 4m
sequence; type 7 (subtype 7a, 7c and 7d sequences), 9, 10 or 11 sequences
of the present invention show type-specific features in this region. The
peptide from amino acid 68 to 78 (V-core region) shows the following
unique sequence for the sequences of the present invention (see FIG. 2):
ARQSDGRSWAQ or ARRSEGRSWAQ as for subtype 1d (SEQ ID NO 107 and 108)
ERRPEGRSWAQ as for subtype 1e (SEQ ID NO 109)
ARRPEGRSWAQ as for subtype 1f (SEQ ID NO 110)
DRRTTGKSWGR as for subtype 2k (SEQ ID NO 111)
DRRATGRSWGR as for subtype 2e (SEQ ID NO 112)
DRRATGKSWGR as for subtype 2f (SEQ ID NO 113)
VRQPTGRSWGQ as for type 9 (SEQ ID NO 114)
VRHQTGRTWAQ as for subtype 7a and 7c (SEQ ID NO 115)
VRQNQGRTWAQ as for subtype 7d (SEQ ID NO 116)
ARRTEGRSWAQ as for type 10 (SEQ ID NO 117)
VRRTTGRXXXX or VRRTTGRTWAQ as for type 11 (SEQ ID NO 118 and 119)
Five type-specific variable regions (V1 to V5) can be identified after
aligning E1 amino acid sequences of the genotypes of the present invention
to the genotypes already known, as shown in FIG. 2.
Region V1 encompasses amino acids 192 to 203, this is the amino-terminal 10
amino acids of the E1 protein. The following unique sequences as shown in
FIG. 2 can be deduced:
HEVRNASGVYHV or HEVRNASGVYHL as for subtype 1d, (SEQ ID NO 120 and 121)
YEVHSTTDGYHV as for subtype 1f (SEQ ID NO 122)
VEVKNTSQAYMA as for subtype 2e (SEQ ID NO 123)
IQVKNNSHFYMA as for subtype 2f (SEQ ID NO 124)
VQVKNTSTMYMA as for subtype 2g (SEQ ID NO 125)
VQVKNTSHSYMV as for subtype 2h (SEQ ID NO 126)
VQVANRSGSYMV as for subtype 2i (SEQ ID NO 127)
VEIKNTXNTYVL or VEIKNTSNTYVL as for subtype 2k (SEQ ID NO 128 and 129)
INYRNVSGIYYV or INYRNTSGIYHV or INYHNTSGIYHI or TNYRNVSGIYHV a for subtype
4k (SEQ ID NO 130, 131, 132 or 133)
QHYRNVSGIYHV as for subtype 4l (SEQ ID NO 134)
IQVKNASGIYHL as for type 9 (SEQ ID NO 135)
AHYTNKSGLYHL as for subtype 7c (SEQ ID NO 136)
LNYANKSGLYHL as for subtype 7d (SEQ ID NO 137)
LEYRNASGLYMV as for type 10 (SEQ ID NO 138)
Region V2 encompasses amino acids 213 to 223. The following unique
sequences can be found in the V2 region as shown in FIG. 2:
IYEMDGMIMHY or IYEMSGMILHA as for subtype 1d, (SEQ ID NO 139 and 140)
VYEAKDIILHT as for subtype 1f (SEQ ID NO 141)
VWQLXDAVLHV as for subtype 2e (SEQ ID NO 142)
VWQLRDAVLHV as for subtype 2f (SEQ ID NO 143)
IWQMQGAVLHV as for subtupe 2g (SEQ ID NO 144)
VWQLKDAVLHV as for subtype 2h (SEQ ID NO 145)
VWQLEEAVLHV as for subtype 2i (SEQ ID NO 146)
TWQLXXAVLHV as for subtype 2k (SEQ ID NO 147)
VYEADHHILHL or VYEADHHILAL or VFEADHHILHL as for subtype 4k (SEQ ID NO 148,
149 and 150)
VYESDHHILHL as for subtype 4l (SEQ ID NO 151)
VFEAETMILHL as for type 9 (SEQ ID NO 152)
VYEAETLILHL as for subtype 7c (SEQ ID NO 153)
VYEANGMILHL as for subtype 7d (SEQ ID NO 154)
VYEAGDIILHL as for type 10. (SEQ ID NO 155)
Region V3 encompasses the amino acids 230 to 242. The following unique V3
region sequences can be deduced from FIG. 2:
VREDNHLRCWMAL or VRENNSSRCWMAL as for subtype 1d (SEQ ID NO 156 and 157)
IREGNISRCWVLP as for subtype 1f (SEQ ID NO 158)
ENSSGRFHCWIPI as for subtype 2e (SEQ ID NO 159)
ERSGNRTFCWTAV as for subtype 2f (SEQ ID NO 160)
ELQGNKSRCWIPV as for subtype 2g (SEQ ID NO 162)
ERHQNQSRCWIPV as for subtype 2h (SEQ ID NO 163)
EWKDNTSRCWIPV as for subtype 2i (SEQ ID NO 164)
EREGNSSRCWIPV as for subtype 2k (SEQ ID NO 165)
VREGNQSRCWVAL or VRTGNQSRCWVAL or VRVGNQSSCWVAL or
VRVGNQSRCWVAL or VKEGNHSRCWVAL as for subtype 4k (SEQ ID NO 166, 167, 168
or 169)
VKTGNTSRCWVAL as for subtype 4l (SEQ ID NO 170)
IKAGNESRCWLPV as for type 9 (SEQ ID NO 171)
VKXXNQSRCWVQA as for subtype 7c (SEQ ID NO 172)
VKTGNLTKCWLSA as for subtype 7d (SEQ ID NO 173)
VRSGNTSRCWIPV as for type 10 (SEQ ID NO 174)
Region V4 encompasses the amino acids 248 to 257. The following unique V4
region sequences can be deduced from FIG. 2:
VKNASVPTAA or VKDANVPTAA as for subtype 1d (SEQ ID NO 175 and 176)
ARIANAPIDE as for subtype 1f (SEQ ID NO 177)
VSKPGALTKG as for subtype 2e (SEQ ID NO 178)
VSRPGALTRG as for subtype 2f (SEQ ID NO 179)
VNQPGALTRG as for subtype 2g (SEQ ID NO 180)
VSQPGALTRG as for subtype 2h (SEQ ID NO 181)
VSQPGALTKG as for subtype 2i (SEQ ID NO 182)
VSRPGALTEG as for subtype 2k (SEQ ID NO 183)
APYIGAPLES or APYTAAPLES as for subtype 4k (SEQ ID NO 184 and 185)
APILSAPLMS as for subtype 4l (SEQ ID NO 186)
VPNSSVPIHG as for type 9 (SEQ ID NO 187)
VPNASTPVTG as for subtype 7c (SEQ ID NO 188)
VQNASVSIRG as for subtype 7d (SEQ ID NO 189)
VKSPCAATAS as for type 10 (SEQ ID NO 190)
Region V5 encompasses the amino acids 294 to 303. The following unique V5
region peptides can be deduced from FIG. 2:
SPRMHHTTQE or SPRLYHTTQE as for subtype 1d (SEQ ID NO 191 and 192)
TSRRHWTVQD as for subtype 1f (SEQ ID NO 193)
APKRHYFVQE as for subtype 2e (SEQ ID NO 194)
SPQYHTFVQE as for subtype 2f (SEQ ID NO 195)
SPQHHNFSQD as for subtype 2g (SEQ ID NO 196)
SPQHHIFVQD as for subtype 2h (SEQ ID NO 197)
SPEHHHFVQD as for subtype 2k (SEQ ID NO 198)
RPRRHWTTQD or RPRRHWTAQD or QPRRHWTTQD or RPRRHWTTQE as for subtype 4k (SEQ
ID NO 199, 200, 201 or 202)
QPRRHWTVQD as for subtype 4l (SEQ ID NO 203)
RPKYHQVTQD as for type 9 (SEQ ID NO 204)
RPRMHQVVQE as for subtype 7c (SEQ ID NO 205)
RPRMYEIAQD as for subtype 7d (SEQ ID NO 206)
RHRQHWTVQD as for type 10 (SEQ ID NO 207)
The above given list of peptides are particularly useful for treatment and
vaccine and diagnostic development.
Also comprised in the present invention is any synthetic peptide (see
below) or polypeptide containing at least an epitope derived from the
above-defined peptides in their peptidic chain. Also comprised within the
present invention is any synthetic peptide or polypeptide comprising at
least 6, 7, 8, or 9 contiguous amino acids derived from the above-defined
peptides in their peptidic chain.
As used herein, `epitope` or `antigenic determinant` means an amino acid
sequence that is immunoreactive. Generally an epitope consists of at least
3 to 4 amino acids, and more usually, consists of at least 5 or 6 amino
acids, sometimes the epitope consists of about 7 to 8, or even about 10
amino acids.
The present invention particularly relates to any peptide (see below) or
polypeptide contained in any of the amino acid sequences as represented in
SEQ ID NO 2, 4, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,
38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72,
74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104 or 106
(see Table 5 and FIG. 3, Examples section).
The present invention also relates to a recombinant polypeptide encoded by
a polynucleic acid as defined above, or a part thereof which is unique to
any of the HCV subtypes or types as defined in Table 5, or an analog
thereof being substantially homologous and biologically equivalent to said
polypeptide.
The present invention also relates to a recombinant expression vector
comprising a polynucleic acid or a part thereof as defined above, operably
linked to prokaryotic, eukaryotic or viral transcription and translation
control elements.
In general said recombinant vector will comprise a vector sequence, an
appropriate prokaryotic, eukaryotic or viral promoter sequence followed by
the nucleotide sequences as defined above, with said recombinant vector
allowing the expression of any one of the polypeptides as defined above in
a prokaryotic, or eukaryotic host or in living mammals when injected as
naked DNA, and more particularly a recombinant vector allowing the
expression of any of the new HCV sequences of the invention spanning
particularly the following amino acid positions:
a polypeptide starting in the region between positions 1 and 10 and ending
at any position in the region between positions 70 and 420, more
particularly a polypeptide spanning positions 1 to 70, 1 to 85, positions
1 to 120, positions 1 to 150, positions 1 to 191, or positions 1 to 200,
for expression of the Core protein, and a polypeptide spanning positions 1
to 263, positions 1 to 326, positions 1 to 383, or positions 1 to 420 for
expression of the Core and E1 protein;
a polypeptide starting at any position in the region between positions 117
and 192, and ending at any position in the region between positions 263
and 420, for expression of E1, or forms that have the hydrophobic region
deleted (positions 264 to 293 plus or minus 8 amino acids);
a polypeptide starting at any position in the region between positions 1556
and 1688, and ending at any position in the region between positions 1739
and 1764, for expression of NS4, more particularly; a polypeptide starting
at position 1658 and ending at position 1711, for expression of NS4a
antigen, and more particularly, a polypeptide starting at position 1712
and ending in the region between positions 1743 and 1972 (for instance
1712-1743, 1712-1764, 1712-1782, 1712-1972, 1712-1782, 1712-1902), for
expression of NS4b antigen or parts thereof.
Any other HCV vector construction known in the art may also be used for the
recombinant polypeptides of the present invention.
Also any of the known purification methods for recombinant proteins may be
used for the production of the recombinant polypeptides of the present
invention, particularly the HCV recombinant polypeptide purification
methods as disclosed in PCT/EP 95/03031 in name of Innogenetics N.V.
The term "vector" may comprise a plasmid, a cosmid, a phage, or a virus or
a transgenic animal. Particularly useful for vaccine development may be
BCG or adenoviral vectors, as well as avipox recombinant viruses.
The present invention also relates to a method for the production of a
recombinant polypeptide as defined above, comprising:
transformation of an appropriate cellular host with a recombinant vector,
in which a polynucleic acid or a part thereof according to as defined
above has been inserted under the control of appropriate regulatory
elements,
culturing said transformed cellular host under conditions enabling the
expression of said insert, and,
harvesting said polypeptide.
The term `recombinantly expressed` used within the context of the present
invention refers to the fact that the proteins of the present invention
are produced by recombinant expression methods be it in prokaryotes, or
lower or higher eukaryotes as discussed in detail below.
The term `lower eukaryote` refers to host cells such as yeast, fungi and
the like. Lower eukaryotes are generally (but not necessarily)
unicellular. Preferred lower eukaryotes are yeasts, particularly species
within Saccharomyces, Schizosaccharomyces, Kluveromyces, Pichia (e.g.
Pichia pastoris), Hansenula (e.g. Hansenula polymoroha), Yarowia,
Schwaniomyces, Schizosaccharomyces, Zygosaccharomyces and the like.
Saccharomyces cerevisiae, S. carlsbergensis and K. lactis are the most
commonly used yeast hosts, and are convenient fungal hosts.
The term `prokaryotes` refers to hosts such as E. coli, Lactobacillus,
Lactococcus, Salmonella, Streptococcus, Bacillus subtilis or Streptomyces.
Also these hosts are contemplated within the present invention.
The term `higher eukaryote` refers to host cells derived from higher
animals, such as mammals, reptiles, insects, and the like. Presently
preferred higher eukaryote host cells are derived from Chinese hamster
(e.g. CHO), monkey (e.g. COS and Vero cells), baby hamster kidney (BHK),
pig kidney (PK15), rabbit kidney 13 cells (RK13), the human osteosarcoma
cell line 143 B, the human cell line HeLa and human hepatoma cell lines
like Hep G2, and insect cell lines (e.g. Spodoptera frugiperda). The host
cells may be provided in suspension or flask cultures, tissue cultures,
organ cultures and the like. Alternatively the host cells may also be
transgenic animals.
The term `recombinant polynucleotide or nucleic acid` intends a
polynucleotide or nucleic acid of genomic, cDNA, semisynthetic, or
synthetic origin which, by virtue of its origin or manipulation: (1) is
not associated with all or a portion of a polynucleotide with which it is
associated in nature, (2) is linked to a polynucleotide other than that to
which it is linked in nature, or (3) does not occur in nature.
The term `recombinant host cells`, `host cells`, `cells`, `cell lines`,
`cell cultures`, and other such terms denoting microorganisms or higher
eukaryotic cell lines cultured as unicellular entities refer to cells
which can be or have been, used as recipients for a recombinant vector or
other transfer polynucleotide, and include the progeny of the original
cell which has been transfected. It is understood that the progeny of a
single parental cell may not necessarily be completely identical in
morphology or in genomic or total DNA complement as the original parent,
due to natural, accidental, or deliberate mutation.
The term `replicon` is any genetic element, e.g., a plasmid, a chromosome,
a virus, a cosmid, etc., that behaves as an autonomous unit of
polynucleotide replication within a cell; i.e., capable of replication
under its own control.
The term `vector` is a replicon further comprising sequences providing
replication and/or expression of a desired open reading frame.
The term `control sequence` refers to polynucleotide sequences which are
necessary to effect the expression of coding sequences to which they are
ligated. The nature of such control sequences differs depending upon the
host organism; in prokaryotes, such control sequences generally include
promoter, ribosomal binding site, splicing sites and terminators; in
eukaryotes, generally, such control sequences include promoters, splicing
sites, terminators and, in some instances, enhancers. The term `control
sequences` is intended to include, at a minimum, all components whose
presence is necessary for expression, and may also include additional
components whose presence is advantageous, for example, leader sequences
which govern secretion.
The term `promoter` is a nucleotide sequence which is comprised of
consensus sequences which allow the binding of RNA polymerase to the DNA
template in a manner such that mRNA production initiates at the normal
transcription initiation site for the adjacent structural gene.
The expression `operably linked` refers to a juxtaposition wherein the
components so described are in a relationship permitting them to function
in their intended manner. A control sequence `operably linked`0 to a
coding sequence is ligated in such a way that expression of the coding
sequence is achieved under conditions compatible with the control
sequences.
The segment of the HCV cDNA encoding the desired sequence inserted into the
vector sequence may be attached to a signal sequence. Said signal sequence
may be that from a non-HCV source, e.g. the IgG or tissue plasminogen
activator (tpa) leader sequence for expression in mammalian cells, or the
.alpha.-mating factor sequence for expression into yeast cells, but
particularly preferred constructs according to the present invention
contain signal sequences appearing in the HCV genome before the respective
start points of the proteins.
A variety of vectors may be used to obtain recombinant expression of HCV
single or specific oligomeric envelope proteins of the present invention.
Lower eukaryotes such as yeasts and glycosylation mutant strains are
typically transformed with plasmids, or are transformed with a recombinant
virus. The vectors may replicate within the host independently, or may
integrate into the host cell genome.
Higher eukaryotes may be transformed with vectors, or may be infected with
a recombinant virus, for example a recombinant vaccinia virus. Techniques
and vectors for the insertion of foreign DNA into vaccinia virus are well
known in the art, and utilize, for example homologous recombination. A
wide variety of viral promoter sequences, possibly terminator sequences
and poly(A)-addition sequences, possibly enhancer sequences and possibly
amplification sequences, all required for the mammalian expression, are
available in the art. Vaccinia is particularly preferred since vaccinia
halts the expression of host cell proteins. Vaccinia is also very much
preferred since it allows the expression of f.i. E1 and E2 proteins of HCV
in cells or individuals which are immunized with the live recombinant
vaccinia virus. For vaccination of humans the avipox and Ankara Modified
Virus (AMV) are particularly useful vectors.
Also known are insect expression transfer vectors derived from baculovirus
Autographa californica nuclear polyhedrosis virus (AcNPV), which is a
helper-independent viral expression vector. Expression vectors derived
from this system usually use the strong viral polyhedrin gene promoter to
drive the expression of heterologous genes. Different vectors as well as
methods for the introduction of heterologous DNA into the desired site of
baculovirus are available to the man skilled in the art for baculovirus
expression. Also different signals for posttranslational modification
recognized by insect cells are known in the art.
The present invention also relates to a host cell transformed with a
recombinant vector as defined above.
The present invention also relates to a method for detecting antibodies to
HCV present in a biological sample, comprising:
(i) contacting the biological sample to be analysed for the presence of HCV
with a polypeptide as defined above,
(ii) detecting the immunological complex formed between said antibodies and
said polypeptide.
The present invention also relates to a method for HCV typing, comprising:
(i) contacting the biological sample to be analysed for the presence of HCV
with a polypeptide as defined above,
(ii) detecting the immunological complex formed between said antibodies and
said polypeptide.
The present invention also relates to a diagnostic kit for use in detecting
the presence of HCV, said kit comprising at least one polypeptide as
defined above, with said polypeptide being preferably bound to a solid
support.
The present invention also relates to a diagnostic kit for HCV typing, said
kit comprising at least one polypeptide as defined above, with said
polypeptide being preferably bound to a solid support.
The present invention also relates to diagnostic kit according as defined
above, said kit comprising a range of said polypeptides which are attached
to specific locations on a solid substrate.
The present invention also relates to a diagnostic kit as defined above,
wherein said solid support is a membrane strip and said polypeptides are
coupled to the membrane in the form of parallel lines.
The immunoassay methods according to the present invention may utilize
antigens from the different domains of the new and unique polypeptide
sequences of the present invention that maintain linear (in case of
peptides) and conformational epitopes (in case of polypeptides) recognized
by antibodies in the sera from individuals infected with HCV. It is within
the scope of the invention to use for instance single or specific
oligomeric antigens, dimeric antigens, as well as combinations of single
or specific oligomeric antigens. The HCVantigens of the present invention
may be employed in virtually any assay format that employs a known antigen
to detect antibodies. Of course, a format that denatures the HCV
conformational epitope should be avoided or adapted. A common feature of
all of these assays is that the antigen is contacted with the body
component suspected of containing HCV antibodies under conditions that
permit the antigen to bind to any such antibody present in the component.
Such conditions will typically be physiologic temperature, pH and ionic
strenght using an excess of antigen. The incubation of the antigen with
the specimen is followed by detection of immune complexes comprised of the
antigen.
Design of the immunoassays is subject to a great deal of variation, and
many formats are known in the art. Protocols may, for example, use solid
supports, or immunoprecipitation. Most assays involve the use of labeled
antibody or polypeptide; the labels may be, for example, enzymatic,
fluorescent, chemiluminescent, radioactive, or dye molecules. Assays which
amplify the signals from the immune complex are also known; examples of
which are assays which utilize biotin and avidin or streptavidin, and
enzyme-labeled and mediated immunoassays, such as ELISA assays.
The immunoassay may be, without limitation, in a heterogeneous or in a
homogeneous format, and of a standard or competitive type. In a
heterogeneous format, the polypeptide is typically bound to a solid matrix
or support to facilitate separation of the sample from the polypeptide
after incubation. Examples of solid supports that can be used are
nitrocellulose (e.g., in membrane or microtiter well form), polyvinyl
chloride (e.g., in sheets or microtiter wells), polystyrene latex (e.g.,
in beads or microtiter plates, polyvinylidine fluoride (known as
Immunolon.TM.), diazotized paper, nylon membranes, activated beads, and
Protein A beads. For example, Dynatech Immunolon.TM. 1 or Immunlon.TM. 2
microtiter plates or 0.25 inch polystyrene beads (Precision Plastic Ball)
can be used in the heterogeneous format. The solid support containing the
antigenic polypeptides is typically washed after separating it from the
test sample, and prior to detection of bound antibodies. Both standard and
competitive formats are know in the art.
In a homogeneous format, the test sample is incubated with the combination
of antigens in solution. For example, it maybe under conditions that will
precipitate any antigen-antibody complexes which are formed. Both standard
and competitive formats for these assays are known in the art.
In a standard format, the amount of HCV antibodies in the antibody-antigen
complexes is directly monitored. This may be accomplished by determining
whether labeled anti-xenogeneic (e.g. anti-human) antibodies which
recognize an epitope on anti-HCV antibodies will bind due to complex
formation. In a competitive format, the amount of HCV antibodies in the
sample is deduced by monitoring the competitive effect on the binding of a
known amount of labeled antibody (or other competing ligand) in the
complex.
Complexes formed comprising anti-HCV antibody (or in the case of
competitive assays, the amount of competing antibody) are detected by any
of a number of known techniques, depending on the format. For example,
unlabeled HCV antibodies in the complex may be detected using a conjugate
of anti-xenogeneic Ig complexed with a label (e.g. an enzyme label).
In an immunoprecipitation or agglutination assay format the reaction
between the HCV antigens and the antibody forms a network that
precipitates from the solution or suspension and forms a visible layer or
film of precipitate. If no anti-HCV antibody is present in the test
specimen, no visible precipitate is formed.
There currently exist three specific types of particle agglutination (PA)
assays. These assays are used for the detection of antibodies to various
antigens when coated to a support. One type of this assay is the
hemagglutination assay using red blood cells (RBCs) that are sensitized by
passively adsorbing antigen (or antibody) to the RBC. The addition of
specific antigen antibodies present in the body component, if any, causes
the RBCs coated with the purified antigen to agglutinate.
To eliminate potential non-specific reactions in the hemagglutination
assay, two artificial carriers may be used instead of RBC in the PA. The
most common of these are latex particles. However, gelatin particles may
also be used. The assays utilizing either of these carriers are based on
passive agglutination of the particles coated with purified antigens.
The HCV antigens of the present invention comprised of conformational
epitopes will typically be packaged in the form of a kit for use in these
immunoassays. The kit will normally contain in separate containers the
native HCV antigen, control antibody formulations (positive and/or
negative), labeled antibody when the assay format requires the same and
signal generating reagents (e.g. enzyme substrate) if the label does not
generate a signal directly. The native HCV antigen may be already bound to
a solid matrix or separate with reagents for binding it to the matrix.
Instructions (e.g. written, tape, CD-ROM, etc.) for carrying out the assay
usually will be included in the kit.
Immunoassays that utilize the native HCV antigen are useful in screening
blood for the preparation of a supply from which potentially infective HCV
is lacking. The method for the preparation of the blood supply comprises
the following steps. Reacting a body component, preferably blood or a
blood component, from the individual donating blood with HCV polypeptides
of the present invention to allow an immunological reaction between HCV
antibodies, if any, and the HCV antigen. Detecting whether anti-HCV
antibody--HCV antigen complexes are formed as a result of the reacting.
Blood contributed to the blood supply is from donors that do not exhibit
antibodies to the native HCV antigens.
In cases of a positive reactivity to the HCV antigen, it is preferable to
repeat the immunoassay to lessen the possibility of false positives. For
example, in the large scale screening of blood for the production of blood
products (e.g. blood transfusion, plasma, Factor VIII, immunoglobulin,
etc.) `screening` tests are typically formatted to increase sensitivity
(to insure no contaminated blood passes) at the expense of specificity;
i.e. the false-positive rate is increased. Thus, it is typical to only
defer for further testing those donors who are `repeatedly reactive`; i.e.
positive in two or more runs of the immunoassay on the donated sample.
However, for confirmation of HCV-positivity, the `confirmation` tests are
typically formatted to increase specificity (to insure that no
false-positive samples are confirmed) at the expense of sensitivity.
The solid phase selected can include polymeric or glass beads,
nitrocellulose, microparticles, microwells of a reaction tray, test tubes
and magnetic beads. The signal generating compound can include an enzyme,
a luminescent compound, a chromogen, a radioactive element and a
chemiluminescent compound. Examples of enzymes include alkaline
phosphatase, horseradish peroxidase and betagalactosidase. Examples of
enhancer compounds include biotin, anti-biotin and avidin. Examples of
enhancer compounds binding members include biotin, anti-biotin and avidin.
In order to block the effects of rheumatoid factor-like substances, the
test sample is subjected to conditions sufficient to block the effect of
rheumatoid factor-like substances. These conditions comprise contacting
the test sample with a quantity of anti-human IgG to form a mixture, and
incubating the mixture for a time and under conditions sufficient to form
a reaction mixture product substantially free of rheumatoid factor-like
substance.
The present invention particularly relates to an immunoassay format in
which the polypeptides (or peptides) of the invention are coupled to a
membrane in the form of parallel lines. This assay format is particularly
advantageous for HCV typing purposes.
The present invention also relates to a pharmaceutical composition
comprising at least one (recombinant) polypeptides as defined above and a
suitable excipient, diluent or carrier.
The present invention also relates to a method of preventing HCV infection,
comprising administering the pharmaceutical composition as defined above
to a mammal in effective amount to stimulate the production of protective
antibody or protective T-cell response.
The present invention relates to the use of a composition as defined above
in a method for preventing HCV infection.
The present invention further relates to a vaccine for immunizing a mammal
against HCV infection, comprising at least one (recombinant) polypeptide
as defined above, in a pharmaceutically acceptable carrier.
The term `immunogenic` refers to the ability of a substance to cause a
humoral and/or cellular response, whether alone or when linked to a
carrier, in the presence or absence of an adjuvant. `Neutralization`
refers to an immune response that blocks the infectivity, either partially
or fully, of an infectious agent. A `vaccine` is an immunogenic
composition capable of eliciting protection against HCV, whether partial
or complete. A vaccine may also be useful for treatment of an individual,
in which case it is called a therapeutic vaccine.
The term `therapeutic` refers to a composition capable of treating HCV
infection. The term `effective amount` refers to an amount of
epitope-bearing polypeptide sufficient to induce an immunogenic response
in the individual to which it is administered, or to otherwise detectably
immunoreact in its intended system (e.g., immunoassay). Preferably, the
effective amount is sufficient to effect treatment, as defined above. The
exact amount necessary will vary according to the application. For vaccine
applications or for the generation of polyclonal antiserum/antibodies, for
example, the effective amount may vary depending on the species, age, and
general condition of the individual, the severity of the condition being
treated, the particular polypeptide selected and its mode of
administration, etc. It is also believed that effective amounts will be
found within a relatively large, non-critical range. An appropriate
effective amount can be readily determined using only routine
experimentation. Preferred ranges of proteins for prophylaxis of HCV
disease are 0.01 to 100 .mu.g/dose, preferably 0.1 to 50 .mu.g/dose.
Several doses may be needed per individual in order to achieve a
sufficient immune response and subsequent protection against HCV disease.
The present invention also relates to a vaccine as defined above,
comprising at least one (recombinant) polypeptide as defined above, with
said polypeptide being unique for at least one of the subtypes or types as
defined above.
Said vaccine compositions may include prophylactic as well as therapeutic
vaccine compositions.
Pharmaceutically acceptable carriers include any carrier that does not
itself induce the production of antibodies harmful to the individual
receiving the composition. Suitable carriers are typically large, slowly
metabolized macromolecules such as proteins, polysaccharides, polylactic
acids, polyglycolic acids, polymeric amino acids, amino acid copolymers;
and inactive virus particles. Such carriers are well known to those of
ordinary skill in the art.
Preferred adjuvants to enhance effectiveness of the composition include,
but are not limited to : aluminim hydroxide (alum),
N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP) as found in U.S. Pat.
No. 4,606,918, N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),
N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn
-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE) and RIBI, which
contains three components extracted from bacteria, monophosphoryl lipid A,
trehalose dimycolate, and cell wall skeleton (MPL+TDM+CWS) in a 2%
squalene/Tween 80 emulsion. Any of the 3 components MPL, TDM or CWS may
also be used alone or combined 2 by 2. Additionally, adjuvants such as
Stimulon (Cambridge Bioscience, Worcester, Ma.)
Immunogenic compositions used as vaccines comprise a `sufficient amount` or
`an immunologically effective amount` of the proteins of the present
invention, as well as any other of the above mentioned components, as
needed. `Immunologically effective amount`, means that the administration
of that amount to an individual, either in a single dose or as part of a
series, is effective for treatment, as defined above. This amount varies
depending upon the health and physical condition of the individual to be
treated, the taxonomic group of individual to be treated (e.g. nonhuman
primate, primate, etc.), the capacity of the individual's immune system to
synthesize antibodies, the degree of protection desired, the formulation
of the vaccine, the treating doctor's assessment of the medical situation,
the strain of infecting HCV, and other relevant factors. It is expected
that the amount will fall in a relatively broad range that can be
determined through routine trials. Usually, the amount will vary from 0.01
to 1000 .mu.g/dose, more particularly from 0.1 to 100 .mu.g/dose.
The proteins of the invention may also serve as vaccine carriers to present
homologous (e.g. T cell epitopes or B cell epitopes from for istance the
core, E1, E2, NS2, NS3, NS4 or NS5 regions) or heterologous (non-HCV)
haptens, in the same manner as Hepatitis B surface antigen (see European
Patent Application 174,444). In this use, envelope proteins provide an
immunogenic carrier capable of stimulating an immune response to haptens
or antigens conjugated to the aggregate. The antigen may be conjugated
either by conventional chemical methods, or may be cloned into the gene
encoding E1 and/or E2 at a location corresponding to a hydrophilic region
of the protein. Such hydrophylic regions include the VI region
(encompassing amino acid positions 191 to 202), the V2 region
(encompassing amino acid positions 213 to 223), the V3 region
(encompassing amino acid positions 230 to 242), the V4 region
(encompassing amino acid positions 230 to 242), the V5 region
(encompassing amino acid positions 294 to 303) and the V6 region
(encompassing amino acid positions 329 to 336). Another useful location
for insertion of haptens is the hydrophobic region (encompassing
approximately amino acid positions 264 to 293). It is shown in the present
invention that this region can be deleted without affecting the reactivity
of the deleted E1 protein with antisera. Therefore, haptens may be
inserted at the site of the deletion.
The immunogenic compositions are conventionally administered parenterally,
typically by injection, for example, subcutaneously or intramuscularly.
Additional formulations suitable for other methods of administration
include oral formulations and suppositories. Dosage treatment may be a
single dose schedule or a multiple dose schedule. The vaccine may be
administered in conjunction with other immunoregulatory agents.
The administration of the immunogen(s) of the present invention may be for
either a prophylactic or therapeutic purpose. When provided
prophylactically, the immunogen(s) is provided in advance of any exposure
to HCV or in advance of any symptom of any symptoms due to HCV infection.
The prophylactic administration of the immunogen serves to prevent or
attenuate any subsequent infection of HCV in a mammal. When provided
therapeutically, the immunogen(s) is provided at (or shortly after) the
onset of the infection or at the onset of any symptom of infection or
disease caused by HCV. The therapeutic administration of the immunogen(s)
serves to attenuate the infection or disease.
In addition to use as a vaccine, the compositions can be used to prepare
antibodies to HCV (E1) proteins. The antibodies can be used directly as
antiviral agents. To prepare antibodies, a host animal is immunized using
the E1 proteins native to the virus particle bound to a carrier as
described above for vaccines. The host serum or plasma is collected
following an appropriate time interval to provide a composition comprising
antibodies reactive with the (E1) protein of the virus particle. The gamma
globulin fraction or the IgG antibodies can be obtained, for example, by
use of saturated ammonium sulfate or DEAE Sephadex, or other techniques
known to those skilled in the art. The antibodies are substantially free
of many of the adverse side effects which may be associated with other
anti-viral agents such as drugs.
The present invention also relates particularly to a peptide corresponding
to an amino acid sequence encoded by at least one of the HCV genomic
sequences as defined above, with said peptide being unique to any of the
HCV subtypes or types as defined in Table 5, and which contains at least
one amino acid differing from any of the known HCV types or subtypes, or
an analog thereof being substantially homologous and biologically
equivalent.
The present invention relates particularly to a peptide comprising at least
one unique epitope of the new sequences of the invention as represented in
SEQ ID NO 1 to 106.
The present invention relates also particularly to a peptide comprising in
its sequence a unique amino acid residue of the invention as defined
above.
The present invention relates particularly to a peptide which is
biotinylated as explained in WO 93/18054.
All the embodiments (immunoassay formats, vaccines, compositions, uses,
etc.) illustrated for the polypeptides of the invention as above also
relate to the peptides of the invention.
The present invention also relates to a method for detecting antibodies to
HCV present in a biological sample, comprising:
(i) contacting the biological sample to be analysed for the presence of HCV
with a peptide as defined above,
(ii) detecting the immunological ccomplex formed between said antibodies
and said peptide.
The present invention also relates to a method for HCV typing, comprising:
(i) contacting the biological sample to be analysed for the presence of HCV
with a peptide as defined above,
(ii) detecting the immunological ccomplex formed between said antibodies
and said peptide.
The present invention also relates to a diagnostic kit for use in detecting
the presence of HCV, said kit comprising at least one peptide as defined
above, with said peptide being preferably bound to a solid support.
The present invention also relates to a diagnostic kit for HCV typing, said
kit comprising at least one peptide as defined above, with said peptide
being preferably bound to a solid support.
The present invention also relates to a diagnostic kit as defined above,
wherein said peptides are selected from the following:
at least one NS4 peptide,
at least one NS4 peptide and at least one Core peptide,
at least one NS4 peptide and at least one Core peptide and at least one E1
peptide,
at least one NS4 peptide and at least one E1 peptide.
The present invention also relates to a diagnostic kit as defined above,
said kit comprising a range of said peptides which are attached to
specific locations on a solid substrate.
The present invention also relates to a diagnostic kit as defined above,
wherein said solid support is a membrane strip and said peptides are
coupled to the membrane in the form of parallel lines.
The present invention also relates to a pharmaceutical composition
comprising at least one as defined above and a suitable excipient, diluent
or carrier.
the present invention also relates to a method of preventing HCV infection,
comprising administering the pharmaceutical composition as defined above
to a mammal in effective amount to stimulate the production of protective
antibody or protective T-cell response.
The present invention also relates to the use of a composition as defined
above in a method for preventing HCV infection.
The present invention also relates to a vaccine for immunizing a mammal
against HCV infection, comprising at least one peptide as defined above,
in a pharmaceutically acceptable carrier.
The present invention relates also to a vaccine as defined above,
comprising at least one peptide as defined above, with said peptide being
unique for at least one of the subtypes or types as defined in Table 5.
The present invention relates to an antibody raised upon immunization with
at least one polypeptide or peptide as defined above, with said antibody
being specifically reactive with any of said polypeptides or peptides, and
with said antibody being preferably a monoclonal antibody.
The monoclonal antibodies of the invention can be produced by any hybridoma
liable to be formed according to classical methods from splenic cells of
an animal, particularly from a mouse or rat, immunized against the HCV
polypeptides according to the invention as defined above on the one hand,
and of cells of a myeloma cell line on the other hand, and to be selected
by the ability of the hybridoma to produce the monoclonal antibodies
recognizing the polypeptides which has been initially used for the
immunization of the animals.
The antibodies involved in the invention can be labelled by an appropriate
label of the enzymatic, fluorescent, or radioactive type.
The monoclonal antibodies according to this preferred embodiment of the
invention may be humanized versions of mouse monoclonal antibodies made by
means of recombinant DNA technology, departing from parts of mouse and/or
human genomic DNA sequences coding for H and L chains or from cDNA clones
coding for H and L chains.
Alternatively the monoclonal antibodies according to this preferred
embodiment of the invention may be human monoclonal antibodies. These
antibodies according to the present embodiment of the invention can also
be derived from human peripheral blood lymphocytes of patients infected
with HCV type 1 subtype 1d, 1e, 1f or 1g, HCV type 2 subtype 2e, 2f, 2g,
2h, 2i, 2k or 2l; HCV type 3, subtype 3g; HCV type 4 subtype 4k, 4l or 4m;
and/or HCV type 7 (subtypes 7a, 7c or 7d), 9, 10 or 11, or vaccinated
against HCV. Such human monoclonal antibodies are prepared, for instance,
by means of human peripheral blood lymphocytes (PBL) repopulation of
severe combined immune deficiency (SCID) mice (for recent review, see
Duchosal et al. 1992) or by screening Eppstein Barr-virus-transformed
lymphocytes of infected or vaccinated individuals for the presence of
reactive B-cells by means of the antigens of the present invention.
The invention also relates to the use of the proteins of the invention,
muteins thereof, or peptides derived therefrom for the selection of
recombinant antibodies by the process of repertoire cloning (Persson et
al., 1991).
Antibodies directed to peptides derived from a certain genotype may be used
either for the detection of such HCV genotypes, or as therapeutic agents.
The present invention relates also to a method for detecting HCV antigens
present in a biological sample, comprising
(i) contacting said biological sample with an antibody as defined above,
(ii) detecting the immune compleexes formed between said HCV antigens and
said antibody.
The present invention relates also to a method for HCV typing, comprising:
(i) contacting said biological sample with an antibody as defined above,
(ii) detecting the immune compleexes formed between said HCV antigens and
said antibody.
The present invention relates also to a diagnostic kit for use in detecting
the presence of HCV, said kit comprising at least one antibody as defined
above, with said antibody being preferably bound to a solid support.
The present invention relates also to a diagnostic kit for HCV typing, said
kit comprising at least one antibody as defined above, with said antibody
being preferably bound to a solid support.
The present invention relates also to a diagnostic kit as defined above,
said kit comprising a range of said antibodies which are attached to
specific locations on a solid substrate.
The present invention relates also to a pharmaceutical composition
comprising at least one antibody as defined above and a suitable
excipient, diluent or carrier.
The present invention relates also to a method of preventing or treating
HCV infection, comprising administering the pharmaceutical composition as
defined above to a mammal in effective amount.
The present invention relates also to the use of a composition as defined
above in a method for preventing or treating HCV infection.
The genotype may also be detected by means of a type-specific antibody as
defined above, which may also linked to any polynucleotide sequence that
can afterwards be amplified by PCR to detect the immune complex formed
(Immuno-PCR, Sano et al., 1992).
Any publications or patent applications referred to herein are incorporated
by reference. The following examples illustrate aspects of the invention
but are in no way intended to limit the scope thereof.
FIGURE LEGENDS
Figure Legends
FIG. 1 Alignment of the nucleotide sequences of the Core/E1 region of some
of the isolates of the newly identified types and subtypes of the present
invention, with other known prototype isolates of subtypes.
FIG. 2 Alignment of the amino acid sequences of the Core/E 1 region of some
of the isolates of the newly identified types and subtypes of the present
invention, with other known prototype isolates of subtypes.
FIG. 3 Nucleotide and amino acid sequences obtained from the new HCV
isolates of the present invention (SEQ ID NO 1 to 106).
FIG. 4 Alignment of the amino acid sequences of the Core/E1 region of some
of the isolates of the newly identified types and subtypes of the present
invention, with other known prototype isolates of subtypes.
FIG. 5 Alignment of the nucleotide sequences of the NS5b region of some of
the isolates of the newly identified types and subtypes of the present
invention, with other known prototype isolates of subtypes.
FIG. 6 Alignment of the amino acid sequences of the NS5b region of some of
the isolates of the newly identified types and subtypes of the present
invention, with other known prototype isolates of subtypes.
Table 5 Overview of the new subtypes and types of the present invention and
the regions sequenced. The subtypes between barckets have been replaced by
the nonbracketed subtypes following the classification of Tokita et al.
(1994).
EXAMPLES
Serum Samples
Serum samples from Cameroonian blood donors (CAM) were screened for HCV
antibodies with Innotest HCV Ab III, and confirmed by INNO-LIA HCV III
(Innogenetics, Antwerp, Belgium). Serum samples from patients with chronic
hepatitis C infection were obtained from various centers in the Benelux
countries (BNL), from France (FR), from Pakistan (PAK), from Egypt (EG),
and from Vietnam (VN).
Samples from the Benelux, Cameroon, France and Vietnam were selected
because of their aberrant reactivities (isolates CAM1078, FR2, FR1, VN4,
VN12, VN13, NE98 and others (see Table 5)).
cPCR, LiPA, Cloning and Sequencing
RNA isolation, cDNA synthesis, PCR, cloning, and LiPA genotyping using
biotinylated 5' UR amplification products were performed as described
(Stuyver et al., 1994c). The 5' UR, the Core/El, and the NS5B PCR products
were used for direct sequencing. The sequence of the universal 5' UR
primers HCPr95, HCPr96, HCPr98, and HCPr29, were described previously
(Stuyver et al. 1993b). The following primers were also described (Stuyver
et al. 1994c): HCPr41, a sense primer for the amplification of the Core
region; HCPr52 and HCPr54 for amplification of the Core/E1 region; and
HCPr206 and HCPr207 for amplification of a 340-bp NS5B region.
Serum samples BNL1, BNL2, BNL3, BNL4, BNL5, BNL6, BNL7, BNL8, BNL9, BNL10,
BNL11, BNL12, CAM1078, FR2, FR16, FR4, FR13, VN13, VN4, VN12, FR1, NE98,
and FR19 were analyzed in the Core/E1 region by direct sequencing. Serum
samples BNL1, BNL2, FR17, CAM1078, FR2, FR16, BNL3, FR4, BNL5, FR13, FR18,
PAK64, BNL8, BNL12, EG81, VN13, VN4, VN12, FR1, NE98, FR14, FR15, and FR19
were also analyzed in the NS5B region by direct sequencing. Partial 5' UR,
Core, E1, and NS5B sequences were obtained. The length of the obtained
sequences is sufficient to classify the obtained sequences into new types
or subtypes, based on the phylogenetic distances to known sequences. The
following sequences could be obtained (nucleotide sequences have
odd-numbered SEQ ID NO., amino acid sequences have even-numbered SEQ ID
NO.): SEQ ID NO 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,
33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67,
69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103
and 105. The amino acid sequences deduced therefrom are given in SEQ ID NO
2, 4, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,
42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76,
78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104 and 106. Table 5
gives an overview of these sequences.
TABLE 5
Type Isolate Nucleotide sequence position
1d BNL1 1-310 (SEQ ID NO. 1) 478-925 (SEQ ID NO. 3)
7932-8271 (SEQ ID NO. 53)
1d BNL2 1-310 (SEQ ID NO. 5) 478-925 (SEQ ID NO. 7)
7932-8271 (SEQ ID NO. 55)
1d FR17
7932-8271 (SEQ ID NO. 57)
1e CAM1078 1-223 (SEQ ID NO. 9) (-238)-414 (SEQ ID NO. 59)
7932-8271 (SEQ ID NO. 61)
1f FR2 1-950 (SEQ ID NO. 11)
7932-8271 (SEQ ID NO. 63)
1g FR16 (-15)-816 (SEQ ID NO. 65)
7932-8271 (SEQ ID NO. 67)
2e BNL3 1-310 (SEQ ID NO. 13) 478-957 (SEQ ID NO. 15)
7932-8271 (SEQ ID NO. 69)
2f FR4 1-957 (SEQ ID NO 17)
7932-8271 (SEQ ID NO. 71)
2g BNL4 478-925 (SEQ ID NO. 19)
2h BNL5 1-310 (SEQ ID NO. 21) 478-925 (SEQ ID NO. 23)
7932-8271 (SEQ ID NO. 73)
2i BNL6 478-833 (SEQ ID NO. 25)
2k FR13 (-238)-957 (SEQ ID NO. 75)
7932-8271 (SEQ ID NO. 77)
2l FR18
7932-8271 (SEQ ID NO. 79)
3g PAK64
7932-8271 (SEQ ID NO. 81)
4k BNL7 1-310 (SEQ ID NO. 27) 478-925 (SEQ ID NO. 29)
4k BNL8 478-925 (SEQ ID NO. 31)
7932-8271 (SEQ ID NO. 83)
4k BNL9 478-925 (SEQ ID NO. 33)
4k BNL10 478-925 (SEQ ID NO. 35)
4k BNL11 478-925 (SEQ ID NO. 37)
4l BNL12 478-925 (SEQ ID NO. 39)
7932-8271 (SEQ ID NO. 85)
4m EG81
7932-8271 (SEQ ID NO. 87)
7a(8b) VN13 1-413 (SEQ ID NO. 45)
7932-8271 (SEQ ID NO. 89)
7c(8a) VN4 1-957 (SEQ ID NO. 43)
7932-8271 (SEQ ID NO. 91)
7d(9a) VN12 1-957 (SEQ ID NO. 47)
7932-8271 (SEQ ID NO. 93)
9a(7a) FR1 1-957 (SEQ ID NO. 41)
7932-8271 (SEQ ID NO. 95)
10a NE98 1-310 (SEQ ID NO. 49) 478-925 (SEQ ID NO. 51)
7932-8271 (SEQ ID NO. 97)
11a FR14
7932-8266 (SEQ ID NO. 99)
11a FR15
7932-8271 (SEQ ID NO. 101)
11a FR19 (-238)-223 (SEQ ID NO. 103)
7932-8271 (SEQ ID NO. 105)
Type Isolate Amino acid sequence position
1d BNL1 1-103 (SEQ ID NO. 2) 159-308 (SEQ ID NO. 4)
2645-2757 (SEQ ID NO. 54
1d BNL2 1-103 (SEQ ID NO. 6) 159-308 (SEQ ID NO. 8)
2645-2757 (SEQ ID NO. 56)
1d FR17
2645-2757 (SEQ ID NO. 58)
1e CAM1078 1-74 (SEQ ID NO. 10) 1-138 (SEQ ID NO. 60)
2645-2757 (SEQ lD NO. 62)
1f FR2 1-316 (SEQ ID NO. 12)
2645-2757 (SEQ ID NO. 64)
1g FR16 1-158 (SEQ ID NO. 66)
2645-2757 (SEQ ID NO. 68)
2e BNL3 1-103 (SEQ ID NO. 14) 159-317 (SEQ ID NO. 16)
2645-2757 (SEQ ID NO. 70)
2f FR4 1-317 (SEQ ID NO. 18)
2645-2757 (SEQ ID NO. 72)
2g BNL4 159-308 (SEQ ID NO. 20)
2h BNL5 1-103 (SEQ ID NO. 22) 159-308 (SEQ ID NO. 24)
2645-2757 (SEQ ID NO. 74)
2i BNL6 159-277 (SEQ ID NO. 26)
2k FR13 1-316 (SEQ ID NO. 76)
2645-2757 (SEQ ID NO. 78)
2l FR18
2645-2757 (SEQ ID NO. 80)
3g PAK64
2645-2757 (SEQ ID NO. 82)
4k BNL7 1-103 (SEQ ID NO. 28) 159-308 (SEQ ID NO. 30)
4k BNL8 159-308 (SEQ ID NO. 32)
2645-2757 (SEQ ID NO. 84)
4k BNL9 159-308 (SEQ ID NO. 34)
4k BNL10 159-308 (SEQ ID NO. 36)
4k BNL11 159-308 (SEQ ID NO. 38)
4l BNL12 159-308 (SEQ ID NO. 40)
2645-2757 (SEQ ID NO. 86)
4m EG81
2645-2757 (SEQ ID NO. 88)
7a(8b) VN13 1-137 (SEQ ID NO. 46)
2645-2757 (SEQ ID NO. 90)
7c(8a) VN4 1-317 (SEQ ID NO. 44)
2645-2757 (SEQ ID NO. 92)
7d(9a) VN12 1-317 (SEQ ID NO. 48)
2645-2757 (SEQ ID NO. 94)
9a(7a) FR1 1-317 (SEQ ID NO. 42)
2645-2757 (SEQ ID NO. 96)
10a NE98 1-103 (SEQ ID NO. 50) 159-308 (SEQ ID NO. 52)
2645-2757 (SEQ ID NO. 98)
11a FR14
2645-2755 (SEQ ID NO. 100)
11a FR15
2645-2757 (SEQ ID NO. 102)
11a FR19 1-74 (SEQ ID NO. 104)
2645-2757 (SEQ ID NO. 106)
Phylogenetic Analysis
Previously published sequences were taken from the EMBL/Genbank database.
Alignments were created using the program HCVALIGN (Stuyver et al. 1994c).
Sequences were presented in a sequential format to the Phylogeny Inference
Package (PHYLIP) version 3.5c (public domain program freely available from
the University of Washington, Seattle, USA). Distance matrices were
produced by DNADIST using the Kimura 2-parameter setting and further
analyzed in NEIGHBOR, using the neighbor-joining setting. The program
DRAWTREE was used to create graphic outputs.
Identification of New Subtypes
These analyses indicated the clustering of BNL1, BNL2, CAM 1078, FR2, FR16,
and FR17 with type 1 isolates, yet neither of these sequences clustered
together with any of the known type 1 subtypes 1a, 1b, or 1c. BNL1, BNL2,
and FR17 clearly clustered together and could be assigned a new type 1
subtype 1d, while CAM1078 could be classified into another new subtype 1e,
FR2 could be classified into another type 1 subtype 1f, and FR16 could be
classified into yet another type 1 subtype 1g. Interestingly, all 3 type
1d isolates (BNL1, BNL2, and FR17) and 1g isolate FR16 were obtained from
patients of Moroccan ethnic origin who resided in Europe.
Another group of isolates showed homology to other type 2 sequences, but
none of the isolates BNL3, FR4, BNL4, BNL5, BNL6, FR13, or FR18 could be
classified into one of the known type 2 subtypes 2a, 2b, 2c (Bukh et al.,
1993), or 2d (Stuyver et al., 1994c). Based on the phylogenetic distances
to other type 2 isolates and to other isolates of the group, each of these
isolates could be classified into a new type 2 subtype. BNL3 was assigned
subtype 2e, FR4 subtype 2f, BNL4 subtype 2g, BNL5 subtype 2h, and BNL6
could be classified into yet another type 2 subtype 2i. If the previously
published isolate HN4 is classified as 2j, FR13 and FR18 may be classified
into new type 2 subtypes 2k and 2l. However, the possibility that FR13 and
FR18 could belong to subtypes 2g or 2i has not yet been ruled out.
Definite classification can be obtained by determining the NS5B sequences
of isolates BNL4 and BNL6, belonging to subtypes 2g and 2i, respectively.
Isolate PAK64 showed homology to type 3 sequences, but could not be
classified into one of the known type 3 subtypes 3a to f. Based on the
phylogenetic distances to other type 3 isolates, PAK64 could be classified
into a new type 3 subtype. PAK64 was assigned subtype 3g. However, the
possibility that PAK64 belongs to a known type 3 subtype can not be
strictly ruled out since only one region of the genome has been sequenced.
Definite classification can be obtained by determining the Core/E1
sequences of isolate PAK64 after amplification with primerHcPr52 and
HcPr54.
Among the Benelux and Egyptian samples that were analyzed, some sequences
clustered with the previously identified type 4 subtypes 4c and 4d.
However, BNL7, BNL8, BNL9, BNL10, BNL11, BNL12, and EG81 clustered into
new subtypes of type 4. Isolates BNL7, BNL8, BNL9, BNL10, and BNL1 1
clustered again separately from BNL12 and EG81 into a new subtype 4k. This
subtype was the predominant subtype in the Benelux countries. BNL12 and
EG81 also segregated into separate subtypes. BNL12 was assigned to another
new subtype 41 and EG81 was assigned to yet another new subtype 4m.
Identification of New HCV Major Types
Isolates FR1, VN4, VN12, VN13, NE98, FR14, FR15, and FR19 did not cluster
with any of the known 6 major types of HCV. VN4, VN12, and VN13 were very
distantly related to genotype 6, but phylogenetic analysis indicated that
these isolates should be assigned new major types. VN13, VN4 and VN12 were
related at the subtype level and assigned type 7a, 7c, and 7d,
respectively. FR1 was not related to any known isolate and was assigned
genotype 9a. NE98 shows a distant relatedness to type 3 sequences, yet
phylogenetic analysis suggested classification into a new major type 10a.
Depending on international guidelines for assigning type and subtype
levels, NE98 may also be classified into an additional type 3 subtype.
FR14, FR15, and FR19 show a very distant relatedness to type 2 sequences,
yet phylogenetic analysis indicated thes isolates to be classified into a
new major type 11, all belonging to the same subtype designated 11a.
Depending on international guidelines for assigning type and subtype
levels, FR14, FR15, and FR19 may also be classified into an additional
type 2 subtype.
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