1. Field of the Invention
The present invention relates to new conformational antibodies directed against HCV and more particularly to monoclonal antibodies. The invention also relates to compositions of particles liable to be recognized by said antibodies, and to pharmaceutical compositions containing them. The invention also relates to HCV enveloped subviral particles or purified HCV enveloped complete viral particles, and the processes for preparing them.
2. Description of the Related Art
Hepatitis C Virus (HCV) infection is a major cause of chronic hepatitis and cirrhosis and may lead to hepatocellular carcinoma. With about 200 million people worldwide chronically infected with HCV, this disease has emerged as a serious global health problem. HCV is an enveloped RNA virus belonging to the genus Hepacivirus of the Flaviridae family. Its genome is a 9.6-kb single-stranded RNA of positive polarity with a 5′ untranslated region (UTR) that functions as an internal ribosome entry site, a single open reading frame encoding a polyprotein of approximately 3,000 amino acids and a 3′UTR (Bartenschlager et al., 2000). This polyprotein is co- and post-translationally cleaved by host cell peptidases to yield the structural proteins including the core protein and the envelope glycoproteins E1 and E2, and by viral proteases to generate the non-structural proteins (NS) 2 to 5B (Bartenschlager et al., 2000). By analogy to related positive-strand RNA viruses, replication occurs by means of a negative-strand RNA intermediate and is catalyzed by the NS proteins, which form a cytoplasmic membrane-associated replicase complex.
The low levels of HCV particles present in patient plasma samples and the lack of a cell culture system supporting efficient HCV replication or particle assembly have hampered the characterization of the glycoproteins associated with the virion. The current knowledge on HCV envelope glycoproteins is based on cell culture transient-expression assays with viral or non viral expression vectors. These studies have shown that the E1 and E2 glycoproteins interact to form complexes (reviewed in Dubuisson, 2000). In the presence of nonionic detergents, two forms of E1E2 complexes are detected: an E1E2 heterodimer stabilized by noncovalent interactions and heterogeneous disulfide-linked aggregates, which are considered to represent misfolded complexes. Previously, envelope glycoprotein-specific antibodies have been obtained by immunization with synthetic peptides or recombinant antigens. A conformation sensitive E2-reactive monoclonal antibody (mAb) (H2), which recognizes noncovalently-linked E1E2 heterodimers considered as the native prebudding form of the HCV glycoprotein heterodimer, however does not react with serum-derived HCV RNA-positive particles (Deleersnyder et al., 1997). Furthermore, WO 92/07001 discloses antibodies which have been prepared by immunization of mice with a preparation of HCV particles extracted from infected chimpanzees, however these antibodies have not been tested on natural HCV particles (i.e. derived from infected patients). Moreover, WO 00/05266 discloses antibodies prepared from infected patients B cells, however these antibodies have been selected according to their ability to bind to the recombinant E2 protein. Therefore, all these antibodies are of limited use, either for diagnostic purposes, or for therapeutic or prophylactic purposes, as they have been produced or selected with unnatural HCV, or parts thereof, and have not been shown to interact with natural HCV particles.
The lack of HCV preparation containing natural enveloped HCV particles in sufficient quantity and concentration, is one of the reason for which antibodies liable to recognize natural HCV particles could not be obtained so far. In fact, low levels of HCV particles in plasma samples have made characterization and visualization of this virus difficult. Previously, it has been shown that virus recovered during the acute phase of infection from the plasma of naturally infected patients has a buoyant density of approximately 1.06 g/ml in sucrose (Hijikata et al., 1993). In contrast, HCV recovered from cell culture after replication in vitro has a buoyant density of 1.12 g/ml in sucrose (Yoshikura et al., 1996). Finally, HCV recovered from chronically infected individuals has a buoyant density of approximately 1.17 g/ml in sucrose (Hijikata et al., 1993). The low density of the serum-derived virus has been ascribed to its association with serum low-density lipoproteins (Thomssen et al., 1992). The high density virus has been shown to be associated with antibody bound to the virus in antigen-antibody complexes (Kanto et al., 1995). In spite of these data, there is as yet no indication on the protein composition of these different HCV populations, and whether low density fractions (<1.0 g/ml) contained envelope, RNA and nucleocapsid as complete virions.