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
2001-02-08
2003-12-30
Housel, James (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...
C424S186100, C424S192100, C424S199100, C424S229100, C435S004000, C435S005000, C435S007100, C435S007900, C435S069100, C435S975000
Reexamination Certificate
active
06669939
ABSTRACT:
The present invention relates to (poly)peptides that are recognized by anti-HHV-8 antibodies of HHV-8 infected patients. By definition, these (poly)peptides shall not comprise naturally occurring HHV-8 proteins. The invention further relates to polymers containing two or more (identical or different) inventory peptides as well as conjugates comprising the inventory peptides and/or polymers thereof. Further we describe mixtures comprising the inventory peptides and/or polymers thereof that are particularly suited for use in procedures to detect anti-HHV-8 antibodies with high sensitivity and specificity. In addition, the present invention relates to a diagnostic kit comprising the inventory peptides, polymers and/or mixtures thereof for the detection of anti-HHV-8 antibodies or diagnosis of HHV-8 infection, respectively.
The human herpesvirus 8 (HHV-8), also known as Kaposi sarcoma associated herpesvirus (KSHV) is the purported etiological agent of Kaposi's sarcoma and certain B cell lymphomas. Kaposi's sarcoma (KS) is the most frequent tumor in AIDS patients, affecting 20-30% of all patients during the course of their HIV infection. In the U.S., the incidence of KS in this risk group is 20,000fold higher than in the general population [1]. In some geographic areas, e.g. in Mediterranean countries or in Africa, the incidence in the general population is significantly higher.
AIDS-associated KS is characterized by an aggressive course and high mortality whereas classical KS is generally relatively indolent and slowly progressing. Other forms of the disease are endemic KS that predominantly affects children and adolescents in Sub-Saharan Africa, and iatrogenic KS in inmnune suppressed transplant recipients.
KS was first described more than a hundred years ago as a relatively rare tumor occurring predominantly in elderly men of Mediterranean or East European origin [2]. The characteristic external manifestations of KS are sharply defined, differently colored (purple, brown, violet or black) nodular lesions of the skin, mostly on the extremities but also in oral mucosa and viscera. Histologically, these lesions consist of long spindle-shaped cells, apparently of endothelial origin, as well as a number of other cell types like fibroblasts, neo-vascular structures, infiltrating leukocytes and extra-vasated red blood cells.
The production of VEGF (vascular endothelial growth factor) is significantly increased in tumor tissue leading to continued angiogenesis and, hence, an extreme vascularization. Already years ago, epidemiological analyses gave hints that an infectious agent might be involved in the development of KS [1, 3]. Using a new PCR technique, Chang and Moore isolated from KS biopsies DNAs from a hitherto unknown human &ggr;-2 herpesvirus [4]. This virus was called KSHV (Kaposi sarcoma associated herpesvirus) or HHV-8 (human herpesvirus 8), respectively. Its 140 kb genome has been recently cloned and sequenced [5].
Since then a series of publications has shown beyond reasonable doubt that all forms of Kaposi's sarcoma are correlated in practically 100% of all cases with the presence of HHV-8 in KS lesions [6-11]. Moreover, PCR detection of HHV-8 is a prognostic marker for later development of Kaposi's sarcoma [12]. Our own work has shown that seroconversion to HHV-8 is detectable on the average already two years before the clinical manifestation of KS.
Most likely HHV-8 is also involved in the pathogenesis of certain lymphoproliferative disorders such as multicentric Castleman's disease (MCD) or primary effusion lymphoma [13, 14]. Some data also hint to a contribution of HHV-8 in interstitial pneumonia and encephalitis [15]. Recently published work suggests that HHV-8 is correlated with multiple myeloma [16]. Although several experimental results are compatible with the hypothesis that HHV-8 may be involved in the pathogenesis of this disease [17-22], a causal relationship is not yet proven. In some geographic regions such as Central Africa, HHV-8 appears to be relatively widespread in the general population [8, 23-25]. Despite a higher incidence of KS in these countries, the presence of an infection with HHV-8 alone appears to be necessary but not sufficient for the development of this disease.
Based on the available viral DNA sequences, several diagnostic tests have been developed that allow a direct or indirect detection of HHV-8. With the help of a relatively simple test, the polymerase chain reaction, even minute quantities of viral DNA can be detected in blood or tissue samples [14, 26-28]. However, for some medical analyses PCR tests are not sensitive enough.
Immunosuppressed organ transplant recipients also have an increased risk of developing Kaposi's sarcoma (up to 5% for kidney transplant recipients); the number of cases varies greatly with geographic origin. In countries of the Near and Middle East, KS is the most common post transplant tumor; worldwide it ranks third. In developed countries (Europe, U.S. or Japan) about 1 to 1.5 m people have an increased risk of getting KS. Blood products for these patients should therefore be unconditionally tested for anti-HHV-8 antibodies as a marker for a possible contamination with HHV-8 in order to keep their infection risk as small as possible. In a broader sense this also applies for other more or less immunocompromised groups, e.g. patients under high dose chemotherapy, dialysis patients, elderly people, neonates etc.
KS predominantly affects people suffering from various forms of immunodeficiency, and is only poorly treatable by chemotherapy, surgery or with ionizing radiation. Presently, there is neither a causative treatment nor a final cure for this disease. One has to consider that immune deficient individuals are exposed to an extraordinary risk to develop Kaposi's sarcoma if they receive HHV-8 contaminated blood products.
For routine diagnostics purposes one mostly uses ELISAs (enzyme-linked immunosorbent assays) because these tests are cheap and suitable for high throughput testing. The detection of HHV-8 specific antibodies indicates prior contact with HHV-8 antigens. This antibody detection can be positive even when the amount of viral DNA (in blood) is below the level of detection in a PCR analysis. Except for ELISAs [29-31], the presence of HHV-8 specific antibodies can also be detected by indirect immunofluorescence assays (IFAs) [25] or Western blots [32, 33].
However, the test procedures that have been described so far are either very time consuming (IFA or Western blot) or not sensitive enough (PCR from blood samples, ELISAs using previously described antigens.
The underlying technical problem for this invention was therefore to define reagents the use of which allows a very sensitive and specific detection of anti-HHV-8 antibodies.
This technical problem is solved by presenting the embodiments detailed in the claims section.
Thus the present invention relates to a (poly)peptide, which is recognized by anti-HHV-8 antibodies in HHV-8 infected patients and is characterized as follows:
(a) it comprises one of the amino acid sequences presented in SEQ ID nos. 1 to 8;
(b) consists of one of the amino acid sequences presented in SEQ ID nos. 1 to 8 or
(c) consists of an amino acid sequence which differs from one of the amino acid sequences described in (a) or (b) by one or several substitutions, deletions and/or insertions; wherein the (poly)peptide is not a naturally occurring HHV-8 protein.
Naturally occurring HHV-8 proteins in the sense of this invention are HHV-8 proteins that have the full-length amino acid sequence and are not degraded. Usually or preferentially, the (poly)peptide is recognized by anti-HHV-8 antibodies in bodily fluids of HHV-8 infected individuals. Methods for the determination of the antigenic property of the (poly)peptide related to the invention as well as methods for the isolation of anti-HH
Haas Jürgen
Schatz Octavian
Biotrin International Properties Limited
Housel James
Li Bao Qun
LandOfFree
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