Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
2000-05-25
2001-06-05
Salimi, Ali R. (Department: 1648)
Chemistry: molecular biology and microbiology
Vector, per se
C435S235100, C435S005000, C536S023720, C536S023100, C536S024100, C514S04400A
Reexamination Certificate
active
06242250
ABSTRACT:
The invention pertains to determined DNA sequences derived from a papillomavirus genome, more particularly DNA recombinants, including vectors, modified by such DNA 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.
The invention pertains more particularly to DNA products of the papillomavirus designated as IP-2 (now re-designated as HPV-33) in the European patent application filed under number 85.402362.9 on Nov. 29, 1965, 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.
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).
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.
Reference is hereafter made to the drawings in which the figs concern respectively
FIGS. 1
a
and
1
b
. Nucleotide sequence of HPV-33. Position 1 on the circular genome corresponds to a “Hpa-like” sequence found by alignment with HPV-6b.
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, EcRV; B2,BqlII; B1, BqlI) 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. 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.
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.
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
DNA Sequence Analysis
The source of HPV-33 sequenced in this study was plasmid p15-5 (1) which consists of a BqlII 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-S, 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° C.).
Computer Analysis.
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
Genomic Arrangement of HPV-33
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.
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 BqlII 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.
Nucleotide Sequence Comparison with HPV-16
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.
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
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 foun
Cole Stewart
Streeck Rolf E.
Finnegan Henderson Farabow Garrett & Dunner
Institut Pasteur
Salimi Ali R.
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