U.S. patent application number 09/832852 was filed with the patent office on 2001-12-06 for determined dna sequences derived from a papillomavirus genome, their uses for in vitro diagnostic purposes and the production of antigenic compositions.
This patent application is currently assigned to INSTITUT PASTEUR. Invention is credited to Cole, Stewart, Streeck, Rolf E..
Application Number | 20010049137 09/832852 |
Document ID | / |
Family ID | 8196291 |
Filed Date | 2001-12-06 |
United States Patent
Application |
20010049137 |
Kind Code |
A1 |
Cole, Stewart ; et
al. |
December 6, 2001 |
Determined DNA sequences derived from a papillomavirus genome,
their uses for in vitro diagnostic purposes and the production of
antigenic compositions
Abstract
The invention concerns DNA fragments derived from the genomic
DNA of HPV-33. These fragments are selected from the group of
fragments extending between the nucleotide extremities defined
hereafter in relation to the nucleotide-numbering in FIGS. 1a and
1b respectively: 76-556 543-864 867-2811 2728-3808 3326-3575
3842-4079 4198-5611 5516-8091 The invention also relates to the use
of these fragments as probes for the detection of HPV in tissue
cultures.
Inventors: |
Cole, Stewart; (Chatillon
Sous Bagneux, FR) ; Streeck, Rolf E.; (La Celle
St-Cloud, FR) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW,
GARRETT and DUNNER, L.L.P.
1300 I Street, N.W.
Washington
DC
20005-3315
US
|
Assignee: |
INSTITUT PASTEUR
Paris
FR
|
Family ID: |
8196291 |
Appl. No.: |
09/832852 |
Filed: |
April 12, 2001 |
Related U.S. Patent Documents
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09832852 |
Apr 12, 2001 |
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09577493 |
May 25, 2000 |
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6242250 |
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09577493 |
May 25, 2000 |
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6107086 |
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09577493 |
May 25, 2000 |
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08789781 |
Jan 28, 1997 |
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08789781 |
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08466711 |
Jun 6, 1995 |
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5648459 |
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08466711 |
Jun 6, 1995 |
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08222569 |
Mar 25, 1994 |
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08222569 |
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08161239 |
Nov 10, 1993 |
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08032694 |
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Current U.S.
Class: |
435/320.1 ;
424/204.1; 435/235.1; 435/5; 435/6.12; 514/44R; 530/350; 530/388.3;
536/23.1; 536/23.72; 536/24.1 |
Current CPC
Class: |
A61P 31/12 20180101;
C12N 7/00 20130101; C12Q 1/708 20130101; A61P 37/04 20180101; C07K
14/005 20130101; C12N 2710/20022 20130101; A61K 39/00 20130101 |
Class at
Publication: |
435/320.1 ;
435/5; 435/6; 530/350; 435/235.1; 536/23.1; 536/23.72; 536/24.1;
514/44; 424/204.1; 530/388.3 |
International
Class: |
C12Q 001/70; C12Q
001/68; C12N 015/86; C07K 014/025; A01N 043/04; A61K 039/12; A61K
031/70; C12N 007/00; C12P 021/08 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 21, 1986 |
GB |
86 400 609 3 |
Mar 20, 1987 |
EP |
PCT/EP87/00158 |
Claims
1. A DNA fragment consisting of an open reading frame, which DNA
fragment is derived from the genomic DNA of HPV-33 and selected
from the group of fragments extending between the nucleotide
extremities defined hereafter in relation to the
nucleotide-numbering in FIGS. 1a and 1b respectively: 76-556
543-864 867-2811 2728-3575 3326-3575 3842-4079 4198-5611
5516-8091
2. A DNA fragment encoding a protein, which DNA fragment is derived
from the genomic DNA of HPV-33 and selected from the group of
fragments extending between the nucleotide extremities defined
hereafter in relation to the nucleotide-numbering in FIGS. 1a and
1b respectively: 109-556 573-864 879-2811 2749-3808 3326-3575
3842-4079 4210-5611 5594-7091
3. The fragment of claims 1 or 2 which is a cloned fragment.
4. A DNA recombinant, replicable in a cell host, particularly
bacteria such as E. coli, which contains an insert containing any
of the fragments defined in claims 1 to 3 fused with a DNA foreign
thereto.
5. The DNA recombinant of claim 4 which is a vector, wherein said
insert is replicable with said vector.
6. The purified peptides obtained from host cells transformed by
the vector of claim 5 and in which said insert has been
expressed.
7. The antibodies against the peptides of the purified peptides of
claim 6.
8. A DNA-hybridization probe for the detection of viral DNA
characterized in that it contains a fragment of any of claims 1 to
3 or is formed of the DNA recombinant of claims 4 or 5, or by a
part thereof.
Description
[0001] The invention pertains to determined DNA sequences derived
from a papillomavirus genome, more particularly DNA recombinants,
including vectors, modified by such ONA sequences in such manner
that, when said DNA recombinants are introduced in suitable host
cells in which said DNA recombinants can be replicated, the said
DNA sequences can be expressed in the form of the corresponding
proteins. The invention further relates to the proteins themselves,
which can be purified and used for the production of immunogenic
compositions.
[0002] The invention pertains more particularly to DNA products of
the papillomavirus designated as IP-2 (now redesignated as HPV-33)
in the European patent application filed under number 85.402362.9
on Nov. 29, 1985, the contents of which are incorporated herein by
reference. A plasmid containing the DNA of said virus has been
deposited at the CNCM ("Collection nationale de Culture de
Micro-Organismes" of the Pasteur Institute of Paris) under number
I-450.
[0003] Papillomaviruses are members of the papovavirus family and
possess a genome of about 7,900 base pairs (bp) consisting of a
covalently closed circular DNA molecule. Human papilloma viruses
(HPV) are classified on the basis of their DNA sequence homology
(6) and nearly 40 types have now been described. Considerable
insight into HPV biology and their involvement in human disease has
been attained by the application of the techniques of molecular
biology. A possible role for HPVs in human cancer was suspected
following the detection of HPV DNA in tumors resulting from the
malignant conversion of genital warts (33). The cloning of two HPV
genomes, HPV-16 and HPV-18 (3, 11) from cervical carcinomas has
further stimulated research in this field of immense socio-economic
importance. These viruses were discovered in more than 70% of the
malignant genital tumors examined and in many others HPV-16 related
sequences were detected (3, 16, 33). Amongst these is HPV-33 which
was recently cloned from an invasive cervical carcinoma using
HPV-16 as a probe under conditions of reduced stringency (1). In
the present study we have determined the DNA sequence of HPV-33 and
describe its relationship to HPV-16. Among the papillomaviruses
HPV-33 is unique as it possesses a 78 bp tandem repeat which
strongly resembles the enhancer of SV40 (4, 14).
[0004] The invention stems from the cloning strategy disclosed
hereafter of the genome of HPV-33 which enabled particular DNA
sequences to be identified, more particularly those providing
hybridization probes, particularly useful for the detection of DNA
of papillomaviruses related to HPV-33 in human tissue, whereby
positive responses can be related to the possible development in
the host of invasive cervical carcinomas.
[0005] Reference is hereafter made to the drawings in which the
figs concern respectively
[0006] FIGS. 1a and 1b. Nucleotide sequence of HPV-33. Position 1
on the circular genome corresponds to a "Hpa-like" sequence found
by alignment with HPV-6b.
[0007] FIG. 2. Distribution of the major reading frames in the
HPV-33 genome. The reading frames were identified by comparison
with other HPV sequences and the stop codons are represdented as
vertical bars. Also indicated are the locations of unique
restriction sites (S, SmaI; E, EcoRV; B2, BglII; B1, BglI) and the
likely polyadenylation signals (PA) for the early and late
transcripts. In addition to these, 6 other potential PA sites
(AATAAA) were detected at positions 862, 1215, 1221, 2666, 5837 and
6239.
[0008] FIG. 3. Principle features of the non-coding region. A
section of the non-coding region from positions 7500 to 114 is
shown. The 78 bp tandem repeats are overlined and those regions
resembling the Z-DNA forming element of the SV-40 enhancer are
indicated. Potential promoter elements are denoted by stars and the
3 copies of the 12 bp palindrome enclosed between two rows of
dots.
[0009] Preferred sequences are those which encode full proteins,
more particularly and respectively the nucleotidic sequences having
the open reading frames referred to in table I hereafter.
[0010] The conditions under which the DNA sequence analysis were
performed are defined under the heading "MATERIALS AND METHODS"
hereafter. The conclusions which were drawn from this sequence
analysis appear under the heading "DISCUSSION".
MATERIALS AND METHODS
[0011] DNA sequence analysis.
[0012] The source of HPV-33 sequenced in this study was plasmid
p15-5 (1) which consists of a BglII linearized HPV-33 genome cloned
in a pBR322 derivative. A library of random DNA fragments (400-800
bp) was prepared in M13mp8 (17) after sonication and end-repair of
p15-5, essentially as described previously (28). DNA sequencing was
performed by the dideoxy chain termination method (19, 20) with the
modifications of Biggin et al. (2). Most of the seQuence was
derived in this way although part of the non-coding region was
found to be absent or under-represented in the M13 library (>300
clones). The sequence of this region was obtained directly from
p15-5 using the method of Smith (24). Briefly, restriction
fragments isolated from 2 "complemenary" M13 clones were used to
prime DNA synthesis on templates prepared from p15-5 which had been
linearized with a restriction enzyme and then treated with
exonuclease III (200 units/pmol DNA for 1 h at 22.degree. C.).
[0013] Computer analysis.
[0014] DNA sequences were compiled and analysed with the programs
of Staden (26, 27) as modified by B. Caudron. Optimal alignments of
DNA or protein sequences were obtained using the algorithm
developed by Wilbur and Lipman (31).
RESULTS AND DISCUSSION
[0015] Genomic Arrangement of HPV-33
[0016] The complete 7909 nucleotide sequence of HPV-33, determined
by the M13 shotgun cloning/dideoxy sequencing approach, is
presented in FIG. 1. On average each position was sequenced 6.5
times. In agreement with the convention for other papillomavirus
sequences the numbering begins at a site resembling the recognition
sequence for HpaI in the non-coding region.
[0017] An analysis of the distribution of nonsense codons (FIG. 2)
shows that, as in all other sequenced papillomaviruses, the 8 major
open reading frames are located on the same strand. Some features
common to HPV-33 and HPV types 1a, 6b and 16 together with the
cottontail rabbit papillomavirus and the prototype bovine
papillomavirus, BPV-1, (5, 7, 8, 13, 21, 22) include the overlap
between the largest open reading frames in the early region, E1 and
E2, and the inclusion of E4 within the section encoding E2.
Interestingly, the BglII site used in the molecular cloning of
HPV-33 is situated within the E1/E2 overlap. Another property
common to all papillomaviruses, except BPV-1, is the overlap
between the L1 and L2 reading frames. Following L1 is the 892 bp
non-coding region which, by analogy with BPV1 (15, 29) undoubtedly
contains the origin of replication and various transcriptional
regulatory elements. The principal characteristics of the HPV-33
genome are summarized in Table 1.
[0018] Nucleotide Sequence Comparison with HPV-16
[0019] HPV-16 is the only other oncogenic papillomavirus, isolated
from tumors of the ano-genital region, which has been completely
sequenced (22). The gross features of HPV-33 resemble those of
HPV-16 except that the E1 reading frame of the latter is
interrupted. All of the coding sequences in HPV-33, except that of
E5, are slightly shorter than their counterparts in HPV-16. This
may contribute to the fact that its non-coding region, between L1
and E6 (FIG. 2), is 76 bp longer thereby keeping the genomes nearly
constant in size.
[0020] When the open reading frames were compared pair-wise (Table
2) it was found that E1, E2, E6, E7, L1 and L2 displayed between
65-75% homology whereas those for E4 and E5 were more divergent
(about 50% homology). These findings confirm the heteroduplex
analysis performed previously (1). A comparative study (8) of
papillomavirus E1 gene products showed that the polypetide consists
of an NH.sub.2-terminal segment whose sequence is highly variable,
and a COOH-terminal domain of well-conserved primary structure. The
longest stretch of perfect sequence homology, 33 nucleotides
(positions 1275-1307, FIG. 1) is found near the 5'-end of the E1
reading frame in a region encoding the variable domain of the
polypeptide. Several other regions of complete identity (19-28
nucleotides) were detected elsewhere in E1, and also in E2, L2 and
L1. As many of these sequences are not found in the genomes of
other HPVs, such as HPV-1a and HPV-6b, this raises the possibility
that the corresponding oligonucleotides could be produced and used
as diagnostic hybridization probes for screening biopsy material
from potentially tumorigenic lesions.
[0021] Potential Gene Products
[0022] The papillomavirus gene products may be divided into those
which are believed to play a purely structural role, L1 and L2, and
those required for viral propagation and persistence. The results
of a comparison of the probable products of the major reading
frames from HPVs-33, 16 and 6b are summarized in Table 2. As
expected there is strong identity between the ocogenic HPVs-33 and
16, particularly for the proposed E1, E6, E7, L2 and L1 proteins.
When conservative substitutions are included the homology between
the two L1 polypeptides increases to 90% suggesting that the
corresponding capsids must be antigenically related. In contrast,
significantly weaker homologies were detected when the analysis was
extended to include the benign genital wart-forming HPV-6b (Table
2). Comparison of the HPV-16 proteins with those of HPV-6b revealed
slightly more homology than was found with HPV-33 suggesting a
closer evolutionary relationship.
[0023] The non-coding Region
[0024] The non-coding region of HPV-33 displays several unique
properties and bears only weak resemblance to its homologue in
HPV-16. Located between the L1 stop codon and including the
putative polyadenylation signal for the late transcripts is a
stretch of 223 bp (positions 7097-7320, FIG. 1) unusually rich in
T+G (79%). Contained within this segment are two copies of a 19 bp
direct repeat (with one mismatch) and 7 copies of the motif TTGTRTR
(where R is A or G). The latter is also found 7 times in the
corresponding region of HPV-16 suggesting that it may represent a
recognition site for proteins involved in replication. It should be
noted that nascent replication forks have been localised in this
regiion of the BPV-1 genome (29) and that the origin of replication
of the Epstein-Barr virus consists of a family of repeated
sequences (32).
[0025] A 12 bp palindrome (ACCG . . . CGGT) that occurs exclusively
in the non-coding region of all papillomavirus genomes examined was
recently reported by Dartmann et al. (9). Three copies were found
in the HPV-33 genome (FIG. 3) and these occupy the same positions
in the non-coding region of HPV-16. A role for the palindrome as a
possible control site for the early promoter was proposed (4, 9,
15) and indirect support is provided by our finding that the
non-coding regions of HPVs, such as HPV-33, do not display the
clustered arrangement of recognition sites for the
promoter-specific, activation factor Sp1(12). This is in direct
contrast to the situation in another papovavirus, SV40 (12,
14).
[0026] The most striking feature of HPV-33 is a perfect 78 bp
tandem repeat located 200 bp after the putative origin of
replication (FIG. 3). No other repeats of this size or sequence
have been described in the genomes of other papillomaviruses. The
presumed early promoter for HPV-33 is located about 300 bp
downstream from the tandem repeat and the characteristic promoter
elements (4) could be identified (FIG. 3). The size, position and
arrangement of the 78 bp repeats in the HPV-33 genome suggest that
they may function as enhancers of viral transcription. Tandem
repeats of 72, 73 and 68 bp have been located near the early
promoter of SV40 (4, 14), in the LTR of moloney murine sarcoma
virus (10), and in the BK virus genome (23) and shown to enhance
transcription from PolII dependent promoters in a cis-active
manner. From mutagenesis of the SV40 enhancer (14, 30) and sequence
comparisons of characterized transcriptional activators a consensus
enhancer sequence was derived. This structure could not be detected
in the 78 bp repeat but a potential Z-DNA forming region was
uncovered. Z-DNA is believed to attract regulatory molecules to
eukaryotic promoters and a Z-DNA antibody binding site has been
demonstrated within the SV40 enhancer (18). The sequence to which
this antibody binds is also found, albeit with a single mismatch,
in the putative HPV-33 enhancer (positions 7520-7527, 7599-7606,
FIGS. 1, 3).
[0027] The proposed HPV-33 enhancer shows no extended sequence
homology to the well-characterized enhancers nor to other
papillomavirus regulatory regions. However, it has recently been
demonstrated that an enhancer-like element is located in the
non-coding region of BPV-1 and that it requires the E2 product for
activation (25). These findings support our proposal that the 78 bp
tandem repeats could have enhancer function and may indicate that
the relatively low homology (Table 2) between the E2 proteins of
HPV-33 and 16 reflects a specificity for the corresponding
enhancer/regulatory regions.
[0028] Tables 1 and 2 which have been referred to in the instant
disclosure follow.
1TABLE 1 Principal features of the HPV-33 genome Open Reading FIRST
STOP Frame START ATG CODON mol. wt. E6 76 109 556 TGA 17 632 E7 543
573 854 TAA 20 825 E1 867 879 2811 TGA 72 387 E2 2728 2749 3808 TAA
40 207 E4 3326 -- 3575 TAG 9 452 E5 3842 -- 4079 TAA 9 385 L2 4198
4210 5161 TAG 50 539 L1 5516 5594 7091 TAA 55 839 a. Calculated
from the first ATG where this exists or from the start of the open
reading frame.
[0029]
2TABLE 2 Comparison of HPV proteins.sup.a HPVs Protein 33v16 33v6b
16v6b E6 65(70) 36(51) 37 E7 61(69) 55(60) 56 E1 61(69) 50(60) 53
E2 53(65) 46(58) 45 E4 52(55) 39(46) 48 E5 40(52) 39(43) 33 L2
64(66) 52(58) 53 L1 81(75) 68(69) 71 .sup.aExpressed as % homology
after alignment with the program of (31). Values in parenthesis
represent % nucleotide sequence homology.
[0030] The invention relates more particularly to sequences
corresponding to the open reading frames of E6, E7, E1, E2, E4, E5,
L2, L1.
[0031] The invention pertains also the uses of these sequences as
hybridization probes, either those which are useful also for the
detection of other papillomaviruses, thus of groups of
papillomaviruses--such as probes containing part or all of the open
reading frames corresponding to L1--or those which are more
virus--specific, i.e. probes containing part or all of the open
reading frame corresponding to.
[0032] It also relates to other probes which detect sub-groups of
papillomaviruses, particularly probes for the detection of viruses
which can be related to major classes of diseases, i.e. viruses
associated with tumors. By way of example of one of said probes one
should mention that which contains the sequence positionned between
nucleotides 1275 and 1307 according to the numbering of the
nucleotides in FIGS. 1A, 1B.
[0033] Needless to say that the invention also pertains to all of
said DNA sequences, when labelled by a suitable label, i.e. a
radioactive enzymatic or immunofluorescent label.
[0034] DNAs derived from the viral genome and which carry
nucleotides modified by a chemical group which can be recognized by
antibodies also form part of the invention. It is well known that
such DNAs can be produced by nick-translation in the presence of
nucleotides modified accordingly. These DNAs form particularly
valuables hybridization probes which, when hybridized to a DNA
preparation containing the complementary strand sought, can be
detected by the above mentioned antibodies.
[0035] The invention also pertains to the diagnostic methods per
se. Suitable methods are examplified hereafter.
[0036] Several hybridization methods may be used. For example, the
spot hybridization method includes, after denaturation of the DNA,
the deposition of an aliquot of the DNA onto film supports
(nitrocellulose or Gene-screenplus), the hybridization of each film
under the usual conditions with the probe, and the detection of the
radioactive hybrid by contact exposition of the hybridized film
onto radiographic film. Another possibility is replicated culture
hyridization which involves agarose gel electrophoresis separation
of the DNA fragments resulting from treatment of the DNA by
restriction enzymes, the transfer of the fragments after alkaline
denaturation onto films (nitrocellulose or Genescreenplus) and
their hybridization under usual conditions with different mixtures
of probes. The formation of radioactive hybrids is detected again
by contact exposition of the hybridization support films onto
radiographic film.
[0037] For instance the probes of the invention can be used for the
detection of the relevant viruses (or DNAs thereof) in preparation
consisting of a biopsy of cells obtained by scraping a lesion, or
of biopsy sections fixed with Carnoy's mixture (ethanol,
chloroform, acetic acid 6:3:1) and included in paraffin.
[0038] The above nucleotide sequences can be inserted in vectors,
to provide modified vectors which, when introduced in the suitable
cell host, are capable of providing for the transcription and,
where appropriate, translation of said DNA sequences to produce the
corresponding proteins which can then be isolated from cellular
extracts of the hosts. Obviously it is within the knowledge of the
man skilled in the art to select the appropriate vectors,
particularly in relation to the host to be transformed therewith.
Vectors consist for instance of plasmids or phages which will be
selected according to their recognized capability of replicating in
the corresponding procaryotic cells (or yeast cells) and of
allowing for the expression of the DNA sequence which they
carry.
[0039] The invention also relates to DNA recombinants containing an
insert consisting of a DNA sequence corresponding to any of the
above-defined open reading frames or of a part thereof, and
suitably engineered to allow for the expression of the insert in
eucaryotic cells, particularly cells of warm-blooded animal.
Suitable DNA recombinants are genetic constructs in which said
insert has been placed under the control of a viral or eucaryotic
promoter recognized by the polymerases of the selected cells and
which further comprise suitable polyadenylation sites downstream of
said insert.
[0040] By way of example, the invention pertains to DNA
recombinants containing any of the above-mentioned-open-reading
inserts placed under the control of a promoter derived from the
genome of the SV40 virus. Such DNA recombinants--or vectors--can be
used for the transformation of higher eucaryotic cells,
particularly cells of mammals (for instance Vero cells). The
invention further pertains to portions of the above identified DNA
sequences which, when inserted in similar vectors, are able to code
for portions of the corresponding proteins which have immunological
properties similar to those encoded by the full nucleotide
sequences mentioned above. The similarity of immunological
properties can be recognized by the capacity of the corresponding
polypeptides produced by the relevant host to be recognized by
antibodies previously formed against the proteins produced by the
cells previously transformed with vectors containing the above
mentioned entire DNA sequences.
[0041] It goes without saying that the invention also pertains to
any nucleotidic sequence related to the preceding ones which may be
obtained at least in part synthetically, and in which the
nucleotides may vary within the constrainsts of the genetic code,
to the extent where these variations do not entail a substantial
modification of the polypeptidic sequences encoded by the
so-modified nucleotidic sequences.
[0042] It already flows from the preceding discussion that the
invention also pertains to the purified proteins or polypeptides
themselves as obtainable by the methods discussed hereabove. These
polypeptides, when produced in a suitable host, can either be
obtained from the cells, for instance after rupturing of their cell
walls, or from the culture medium of said cells when excreted in
said cell medium, depending on the cell DNA recombinant system
which is used. The polypeptide obtained can then be purified by
resorting to usual purification procedures. It should be understood
that purified in the instant context means a level of purity such
that, when electrophoresed in SDS-PAGE, the purified proteins yield
a single detectable band, say by Western blot.
[0043] The viral proteins obtained, more particularly the
structural proteins, for instance as a result of the expression of
said DNA sequences in E coli, can be used for the in vitro
detection of antibodies against papillomavirus likely to be
detected in tissue samples of patients possibly infected with
papillomavirus. of particular relevance are the genetically
engineered proteins having the peptidic sequences which can be
deduced from the L1 and L2 open reading frames. Another peptide of
interest is the E6 protein (E6 star), the synthesis of which can be
induced by splicing and which encoded by a nucleotidic sequence
located between nucleotides 229 (donor site) and 404 (acceptor
site) of the HPV 33 sequence (see more particularly FIG. 1A), which
sites also define the putative splicing sites in the E6 open
reading frame of HPV 33. Reference may be had to the publication of
Schneider-Gardicke and Schwartz, Embo. J., 5, 2285-2292, as
concerns the conditions of the production of such proteins.
[0044] These purified polypeptides can in turn be used for the
production of corresponding antibodies which can be used for
diagnosing in vitro the presence of viral polypeptides in a
biological fluid, particularly in a serum or tissue culture of a
patient. Like in the preceding instance, the invention relates to
portions of the above defined polypeptides, particularly those
which are recognized by the same antibodies or to the contrary are
able to elicit in vivo the production of antibodies recognizing the
complete proteins.
[0045] It must be understood that the inventions relates also
specifically to the particular peptides encoded by the DNA regions
specifically referred to in the preceding disclosure and which have
been found of particular interest.
[0046] The invention further concerns host cells transformed with
DNA recombinants containing nucleotidic sequences directing the
expression of the different peptides mentioned hereabove, and
effectively capable to produce said peptides when cultured in an
appropriate culture medium.
[0047] The invention finally also pertains more particularly to the
antibodies themselves which can be obtained from an animal, such as
rabbit, immunized in standard manner with said purified
polypeptides and/or from hybridomas previously prepared also in any
known manner. Of particular inerest are the antibodies (polyclonal
and monoclonal antibodies) directed against the strutural proteins.
These antibodies are useful for the detection of viral infection.
The antibodies which recognize the L1, L2 and E6 proteins of HPV-33
are of particular significance. Antibodies specific of L2 provide
diagnostic tools for the in vitro detection of specific viruses
sharing with HPV-33 a sequence encoding a similar L2 protein.
Antibodies specific to L1 are useful for the detection of the
groups of viruses, to which HPV-33 belongs. Antibodies specific to
the E6 protein are useful for the detection of the oncogenic
character of the virus causing the abovesaid viral infection.
[0048] The invention also relates to intergenic sequences of
particular interest, particular the 78 bp sequence. This sequence
is of particular interest as a possible insert in eucaryotic
vectors, particularly in a position upstream of the promoter and
downstream of the site at which transcription of the gene or
nucleotide sequence the transcription of which is sought is
initiated in the relevant host.
[0049] All documents referred to herein are incorporated herein by
reference. Particularly these documents can be referred to as
concerns the definition of expressions used in this application
where appropriate. As such they form part of the present
disclosure.
BIBLIOGRAPHY
[0050] 1. Beaudenon, S.
[0051] 2. Biggin, M. D., T. J. Gibson, and G. F. Hong. 1983 Buffer
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