Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
2000-05-05
2003-12-30
Wortman, Donna C. (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S007800, C435S007930
Reexamination Certificate
active
06670114
ABSTRACT:
FIELD OF THE INVENTION
The present invention is based on the development of an efficient infection system for HCV, and on the finding that the human proteins annexin V, tubulin and apolipoprotein B bind to the hepatitis C virus envelope proteins E1 and/or E2 and concerns the usage of these human proteins to diagnose and treat an infection with hepatitis C virus. The present invention also relates to the usage of the latter proteins to enrich HCV envelope proteins and to molecules which inhibit binding of HCV to these human proteins, as well as vaccines employing the E1 and/or E2 binding domains.
BACKGROUND OF THE INVENTION
Hepatitis C virus (HCV) infection is a major health problem in both developed and developing countries. It is estimated that about 1 to 5% of the world population is affected by the virus, amounting up to 175 million chronic infections worldwide. HCV infection appears to be the most important cause of transfusion-associated hepatitis and frequently progresses to chronic liver damage. Moreover, there is evidence implicating HCV in induction of hepatocellular carcinoma. Consequently, the demand for reliable diagnostic methods and effective therapeutic measures is high. Also sensitive and specific screening methods for HCV-contaminated blood-products and improved methods to culture HCV are needed.
HCV is a positive stranded RNA virus of about 9,8 kilobases which code for at least three structural and at least six non-structural proteins. The structural proteins have not yet been functionally assigned, but are thought to consist of a single core protein and two envelope proteins E1 and E2. The E1 protein consists of 192 amino acids and contains 5 to 6 N-glycosylation sites, depending on the HCV genotype, whereas the E2 protein consists of 363 to 370 amino acids and contains up to 11 N-glycosylation sites, depending on the HCV genotype (for review see Maertens and Stuyver, 1997). The latter envelope proteins have been produced by recombinant techniques using
Escherichia coli
, baculovirus, yeast and mammalian expression systems. The usage of an expression system in higher eukaryotes and especially in mammalian cell culture leads to envelope proteins of superior quality, i.e. they are effectively recognized by antibodies recovered from HCV patients (Maertens et al., 1994, de Martynoff et al., 1996).
Standardized infections of live viruses are a prerequisite for studying the binding parameters of HCV to eukaryotic cells. Controllable infection of eukaryotic cells by HCV, however, poses a problem. As a partial solution to this problem, a Daudi cell line was selected, which was supporting productive infection for HCV (Shimizu et al., 1996). However, the inocula for infection gave variable results in Molt-4 cells and even in Daudi cells. Consequently, comparative studies, e.g. on the development of drugs interfering with the interaction of HCV with its target eukaryotic cell are troublesome. Therefore, there is an urgent need for a protocol, which guarantees efficient and reliable infections of eukaryotic cells by HCV.
The HCV envelope proteins E1 and E2 interact with each other to form hetero-oligomeric complexes. Although the exact role of these HCV envelope proteins has not been elucidated yet, it has been suggested that they are responsible for binding of the virus to target cells. Indeed, binding of E2, mostly involving the highly variable amino terminus of E2 (i.e. the hyper variable region I), to target cells has been documented by several authors (Farci et al., 1996; Shimizu et al., 1994; Zibert et al., 1995 and Rosa et al., 1996). Furthermore, several host proteins have been shown to bind to one or both envelope proteins. For example, the chaperone protein calnexin has been shown to interact with both E1 and E2 and, by doing so, to support correct folding of both envelope proteins (Deleersnyder et al., 1997). Also lactoferrin, a protein mainly found in milk, has been demonstrated to bind to both envelope proteins (Yi et al., 1997b). However, the role of this interaction is unclear. A 24 kDa plasma membrane protein has been described which binds specifically to E2 (WO 97/09349 to Abrignani). The latter protein has been suggested to be a cellular receptor for HCV. Also the mannose receptor on hepatic endothelial cells and macrophages and the asialoglycoprotein receptor on hepatocytes have been suggested to function as receptors for HCV via the E1 and E2 proteins (WO92/08734 to Raiston et al.). In addition, RNA of HCV in serum of patients is often found associated with the LDL-fraction of serum (Thomsson et al., 1992 & 1993; Agnello et al., 1996 &1997). The nature of this finding, has not been unravelled yet.
Taken together, several HCV-binding proteins have been described in the literature. However, no prior art exists regarding the binding of the human proteins annexin V, apolipoproteinB, or tubulin to the hepatitis C virus envelope proteins E1 and/or E2. Neither has the interaction between HCV and LDL been further unravelled with regard to the components of LDL involved, ie protein, lipid or sugar, nor with regard to the regions of the HCV proteins involved, ie the envelope proteins E1 and/or E2.
Annexin V (also termed endonexin II, placental anticoagulant protein, PP4 or lipocortin V) is a member of the family of structurally closely related Ca
2+
-dependent phospholipid-binding proteins, known as annexins, which have molecular weights between 32 and 67 kDa (Klee, 1988; Zaks & Creutz, 1990). Annexin V is found in various tissues such as liver, spleen, lung, intestine and placenta (Walker et al., 1990). The protein has been described to bind, in a Ca
2+
-dependent manner, to placental membranes (Haigler et al., 1987) and to inhibit blood coagulation (Grundman et al., 1988) and phospholipase A2 activity in vitro (Pepinsky et al., 1988). Other investigators have demonstrated that annexin V behaves like an integral membrane protein and forms calcium-selective cation channels (Rojas et al., 1990; Bianchi et al., 1992).
We have recently shown that annexin V, present on human liver plasma membranes, specifically binds to “small” HBsAg, one of the envelope glycoproteins of hepatitis B virus (HBV), in a Ca
24+
-dependent manner (Hertogs et al., 1993; WO 94/01554). The receptor-ligand relationship between HBsAg and annexin V is further supported by the observation that rabbits, immunized with native human liver annexin V or recombinant annexin V, or chickens, immunized with F(ab')
2
-fragments of rabbit anti-annexin V IgG, spontaneously develop anti-idiotypic antibodies (Ab2) which specifically recognize HBsAg (Hertogs et al., 1994). Since HCV is an RNA virus belonging to the family of flaviviridae, and HBV is a DNA virus belonging to the family of hepadnaviruses, the binding of annexin to E1 or E2 of HCV was completely unexpected.
Tubulin is a soluble protein found in most eukaryotic cells and is the principal protein subunit of microtubules in the cell. Microtubules are the principal components of mitotic and meiotic spindles and of the axons of neuronal cells. Microtubules also participate in several aspects of intracellular transport, in maintenance of various cell surface properties such as receptor capping and they establish overall cell shape and internal cytoplasmic architecture. Tubulin extracted from neurons is a dimer of about 100 kDa. each dimer being composed of two polypeptides &agr;-tubulin (50 kDa) and &bgr;-tubulin (50 kDa), which have closely related amino acid sequences (Alberts et al., 1983).
Tubulin has been implicated in the transcription of Sendai virus (Takagi et al, 1996) and has been shown to be involved during intracellular transport of herpes simplex virus type 1 (Hammonds et al., 1996) and tobacco mosaic virus (McLean et al., 1995). Tubulin appears also to interact with the matrix protein of vesicular stomatitis virus (Melki et al., 1994).
Apolipoprotein B (ApoB) represents the main protein component of the low-density lipoproteins (LDL). Besides its lipid-carrier property apoB is involved in the secretion into the pl
Depla Erik
Maertens Geert
N.V. Innogenetics
Wortman Donna C.
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