Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Virus or component thereof
Reexamination Certificate
1993-12-10
2001-11-27
Scheiner, Laurie (Department: 1648)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Virus or component thereof
C424S139100, C424S141100, C424S147100, C424S186100, C435S005000, C435S339000, C530S388300, C530S389400
Reexamination Certificate
active
06322794
ABSTRACT:
The invention relates to seroreactive regions on proteins E1, E6 and E7 of human papillomavirus (HPV) 18.
The invention also relates to vaccines which contain peptides which embrace amino-acid sequences of the seroreactive regions of the said virus proteins and to diagnostic kits which contain the said peptides.
HPV18 is a specific type of human papillomavirus which was described for the first time in Proc. Natl. Acad. Sci., USA 80, 3813-3815 (1983).
The DNA sequence and the organization of the viral genome of HPV18 were published in Virology 145, 181-185 (1985).
HPV18 induces not only benign lesions of the anogenital tract but also malignant tumors of the neck of the uterus, of the penis and of the vagina. However, HPV18 is also found in genital scrapings from clinically symptom-free individuals. To date little is known about the immune response to infection by HPV18 and other papillomaviruses.
In initial experiments, human sera from STD patients, from patients suffering from cervical tumors and from healthy individuals were tested for the presence of antibodies directed against viral proteins. These viral proteins were expressed as fusion proteins covalently bonded to various procaryotic peptides via their N terminus. Fusion proteins of this type were then used as antigens in Western blot experiments. However, this test is relatively time-consuming and complicated and can be carried out only with great expense, so that it appears unsuitable for quantitative analysis of large quantities of human serum. In addition, this test is not very specific because the various papillomavirus types are also related in respect of their protein core, and thus a cross-reaction of antibodies with proteins or fusion proteins from different papillomavirus types cannot be ruled out.
The object of the present invention is therefore to identify viral structures of HPV18 which are suitable as aids in the prophylaxis, the diagnosis and the therapy of HPV18-induced diseases in humans. Knowledge about such structures (protein domains) is a prerequisite for the establishment of a test with which large quantities of human blood serum can be examined for the presence of specific HPVs.
Thus the present invention embraces a seroreactive epitope on the El protein of HPV18 with the following amino-acid sequence
TENSPLGERLEVDTELSPRLQEISLNS (SEQ ID No:1)
and seroreactive epitopes on the E6 protein of HPV18 with one of the following amino-acid sequences
I. DPTRRPYKLPDLCTELNTSLQDIEITCVYCKT (SEQ ID No:2)
II. MARFEDPTRRPYKL (SEQ ID No:3)
III. AACHKCIDFYSRIRELRHYSDSVYGDTLEKLT (SEQ ID No:4) and seroreactive epitopes on the E7 protein of HPV18 with one of the following amino-acid sequences
I. VLHLEPQNEIPVDLLCHEQLSDSEEENDEIDGVNHQHLPARRAEPQRH (SEQ ID No:5) and
II. IDGVNHQHLPARR (SEQ ID No:6).
The invention also embraces peptides which contain either one or more than one amino-acid sequence(s) according to the invention of the abovementioned seroreactive epitopes.
The invention also embraces vaccines which are based on peptides which contain one or more amino-acid sequence(s) of the abovementioned seroreactive epitopes of the proteins of HPV18.
Specific antibodies against HPV18 E1, E6 and E7 proteins can be detected in patients' sera using a diagnostic kit according to the invention. This kit contains the peptides according to the invention.
Moreover, it is also possible, with a view to prophylaxis, for the specific viral proteins which contain the seroreactive regions to be identified in blood serum in good time using polyclonal antibodies or monoclonal antibodies which are directed against these regions. Accordingly, this invention also embraces a diagnostic kit which contains polyclonal or monoclonal antibodies which are specifically directed against the seroreactive regions of HPV18.
The following mutually independent methods were used to identify the seroreactive epitopes:
A. Screening of a shotgun expression bank: the HPV18 DNA cloned in the bacterial plasmid vector pSP65 was converted into fragments with an average size of 100 base-pairs by ultrasonic shearing and subsequent DNase treatment. The ends of these fragments were filled in with T4 DNA polymerase and ligated into the phage expression vector fuse 1. Fuse 1 is derived from the bacteriophage fd and is described in Science 228, 1315-1317 (1985). The phages were plated out with
Escherichia coli
K91, replicas were made on nitrocellulose filters, and the filters were incubated with suitable polyclonal rabbit sera. Positive clones were isolated in several singling-out steps, and the immunoreactive peptide sequences were identified by DNA sequencing.
B. Peptide overlapping
127 overlapping peptides which correspond to short segments of the HPV18 E6 and E7 proteins were synthesized on polyethylene pins using Fmoc chemistry (Proc. Natl. Acad. Sci., 82, 178 (1985)). The protein sequence of the E6 and E7 proteins was divided into
10
mers which coincide in 8 amino acids with the next peptide. The peptides were assayed by ELISA in the appropriate antisera.
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Bleul Conrad
Gissmann Lutz
Muller Martin
Dade Behring Marburg GmbH
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Scheiner Laurie
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