Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Amino acid sequence disclosed in whole or in part; or...
Reexamination Certificate
1998-07-08
2001-01-23
Salimi, Ali (Department: 1648)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Amino acid sequence disclosed in whole or in part; or...
C424S204100, C424S229100, C424S230100, C435S005000, C435S007100, C435S007920, C435S007940, C435S235100, C530S350000, C530S300000, C536S023720
Reexamination Certificate
active
06177080
ABSTRACT:
Kaposi sarcoma is a vascular tumor of the skin which is now of great clinical and epidemiological importance. Originally, Kaposi sarcoma occurred rarely and predominantly in the older male population of south and south east Europe (especially Italy and Greece).
With the spread of human immunodeficiency virus (HIV), Kaposi sarcoma has achieved considerable importance and now represents one of the commonest manifestations in AIDS patients.
In recent years, a herpes virus has been discovered to occur in Kaposi sarcoma tissue from AIDS patients in a very high percentage. This virus has been referred to as Kaposi sarcoma-associated herpes virus (KSHV) or HHV-8. Russo et al. have recently published the nucleotide sequence of Kaposi sarcoma-associated herpes virus [
Proc.Natl.Acad.Sci., USA
, Vol. 93 (December 1996), pages 14862-14867].
The nucleotide sequence of KSHV shows noticeable sequence homologies with known gamma-herpesviruses such as, for example, the herpesvirus Saimiri (HVS), which occurs in monkeys, and the Epstein-Barr virus (EBV). Taxonomically, the virus is included in the Rhadinoviridae group.
The genome of the virus comprises about 165 kilobases, and Russo has identified 81 open reading frames within this sequence, of which 66 are homologous with open reading frames of the herpes virus Saimiri.
It has emerged that the genome of KSHV can also be detected in AIDS-independent classical and endemic forms of Kaposi sarcoma, as well as in other rare tumors such as body cavity based lymphoma (BCBL) and Castleman's disease (CD).
The mechanism of transmission of KSHV has not yet been completely elucidated. Since the virus can be detected in sperm, there is unlikely to be any doubt about sexual transmission in homosexual men. However, there must also be another transmission route in endemic areas.
It has not yet been possible to infect cells with KSHV in vitro reliably and with adequate efficiency. There are B-cell culture systems, for example BC-1 and BCBL-1, in which the virus shows latent persistence, and the lytic replication cycle can be stimulated with a phorbol ester, namely tetradecanoylphorbol acetate (TPA).
For constructing the gene bank, Russo et al. used the BC-1 cell line which is coinfected both with KSHV and with Epstein-Barr virus.
By contrast, according to the invention, the BCBL-1 cell line which is infected only with KSHV has been used. When this cell line is induced with TPA, KSHV-specific antigen expression takes place in the cell culture, and the antigens expressed in this way have been employed to provide the polypeptides according to the invention. The lysates of induced BCBL-1 cells were initially investigated.
The cell culture-dependent Western blot and immunofluorescence analyses establish 95% sensitivity with the sera from AIDS patients with Kaposi sarcoma. It was possible to show by these analyses that the seroprevalence of KSHV in homosexual, HIV-positive men is significantly greater than in HIV-positive hemophilia patients, so that it can be assumed that the virus is transmitted by a sexual route. The seroprevalence in the normal population is subject to wide regional variations. The seroprevalence in the USA and northern Europe is below 0.5%, whereas in Italy 5% and in Uganda 50% of the blood samples from healthy blood donors show antibodies against KSHV. It was also possible to show that about 6 to 75 months before the appearance of a Kaposi sarcoma it was possible to detect seroconversion of KSHV antibodies, and a prospective statement about the clinical manifestation of Kaposi sarcoma was possible. Thus detection of KSHV-specific antibodies represents a suitable diagnostic marker for most experts. Because of the considerable technical complexity and the lack of standardization, the existing serological test methods are suitable only for special diagnostic laboratories.
The provision of a recombinant capsid protein encoded by open reading frame 65 (ORF65) has been described. It was possible to show that the C-terminal part of the protein (AA 86-170) has the same serological reactivity as the full-length protein [Simpson et al.,
Lancet
, Vol. 348 (October 1996), pages 11033-11038]. Lin et al. also used recombinant OR65 antigen in serological tests for detecting antibodies against Kaposi sarcoma-associated herpes virus. In this case, this is the small viral capsid antigen (sVCA) [
Journal of Virology
(April 1997), pages 3069-3076] which represents the homologous protein to BFRF3 of EBV.
It is therefore one object of the present invention to provide further polypeptides which are suitable for detecting KSHV in standardized and automated methods. Characterization and identification of suitable immunologically reactive antigens is necessary for immunoblots or enzyme-linked immunosorbent assays (ELISA).
The polypeptides according to the invention are encoded by a reading frame which has not previously been identified and is referred to according to the invention as ORF K8.1. This reading frame was identified in the following way:
It was possible to detect in Western blot analyses, with BCBL-1 cells stimulated to lytic KSHV replication with TPA, using sera from particular patients (HIV-positive with Kaposi sarcoma) a protein double band with a molecular weight of about 35-37 kilodaltons. Deglycosilation experiments were carried out to characterize the protein (gp35/37). It emerged from these that the proteins with a migration behavior of 35 and 37 kDa in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) can be converted by deglycosylating enzymes into one protein band with 30 kDa. It was concluded from this that the protein must be a viral glycoprotein which is present in two glycosilated forms in the KSHV-infected cell.
Since the sequence of the KSHV from the BC-1 cell has now been determined, this sequence was screened using various screening criteria to identify a suitable open reading frame. The specific criteria used for this do not comply with the usual rules. Initial screening was only for open reading frames having a start codon (ATG). It was not a requirement that a TATA box precede the start of transcription. It was possible in this way to find open reading frames with start codons which did not comply with the usual Kozack rules. Among the reading frames found in this way, only those reading frames coding for a protein with a molecular weight between 15 and 45 kDa were taken into account. In addition, the sequences selected from the reading frames found in this way were those having an N-glycosilation site. The potential open reading frames found in this way were also limited by the requirement for the presence of a signal peptide. Five of the initially found 41 reading frames met the abovementioned conditions, namely the open reading frames ORF47 (glycoprotein L); K2 (IL6-homologous); ORF70 (thymidylate synthase); K1 and K8.1. ORF70 was not taken into further account subsequently. The coding sequence of ORF47, K2, K1 and K8.1 was in each case cloned into the expression vector pQE9 (from Qiagen), and the proteins were expressed as histidine fusion proteins in
E.coli
. It was possible due to the histidine fusion protein content to purify the proteins using nickel chelate agarose and employ them in Western blot tests. The sera used for this were those which also reacted with the protein gp35/37 in the natural Western blot. Whereas the proteins of reading frames ORF47, K2, K1 showed no reactivity whatsoever, all the sera reacted with the recombinant K8.1 protein. Subsequent reexamination of the publication by Russo et al. revealed that the reading frame ORF K8.1 used according to the invention is not mentioned.
In order to demonstrate that the protein gp35/37 in the natural Western blot with BCBL-1-induced cells is identical to the recombinant protein prepared with the aid of the reading frame ORF K8.1 according to the invention, antibodies from sera from KS-positive patients were bound by means of preparative Western blot analysis to recombinant gp35/37. Antibodies recovered by subsequent elution were t
Albrecht Jens-Christian
Fleckenstein Bernhard
Lang Dieter
Neipel Frank
Biotest AG
Heller Ehrman White & McAuliffe LLP
Salimi Ali
Seidman Stephanie
LandOfFree
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