Title:  Complex of lipo-viro-particles, method of preparation and applications

United States Patent:  6,548,295

Issued:  April 15, 2003

Inventors:  Andre; Patrice (Lyons, FR); Lotteau; Vincent (Vourles, FR); Paranhos-Baccala; Glaucia (Lyons, FR); Komurian-Pradel; Florence (Poleymieux, FR)

Assignee:  Bio Merieux (Mercy l'Etoile, FR); Institut National de la Sante et de Recherche Medicale (Paris, FR)

Appl. No.:  917915

Filed:  July 31, 2001

The complex consists of LVPs associated with human immunoglobulins having a density of less than 1.063 g/ml. It may be obtained by a method according to which a plasma or serum sample taken from a patient infected with HCV is made available, the LVPs are separated from the said sample by centrifugation according to their density, and the LVPs associated with human immunoglobulins are separated using protein A, anti-human immunoglobulins or any other molecule capable of binding human immunoglobulins.

DETAILED DESCRIPTION OF THE INVENTION

The invention also relates to a method for preparing the said LVPs associated with human immunoglobulins (LVP/Ig) complex which consists in carrying out a separation by gradient centrifugation from a plasma or serum sample from a patient for the production of a fraction of the LVP/Ig complex having a density of less than 1.063 g/ml and preferably of between 1.0063 and 1.063 g/ml, and then a purification of the fraction of the said complex by protein A or human anti-immunoglobulins or by any other molecule capable of binding human immunoglobulins (coupled to a support, such as beads or sepharose). This method makes it possible "to enrich" the initial sample with LVP/Ig complex while eliminating the lipoproteins not belonging to the LVP/Ig complex.

The LVP/Ig complex obtained, preferably purified according to the mode of preparation of the invention can be used for carrying out a method for the in vitro culture of the HCV virus. Indeed, a large number of cells possess, at their surface, a receptor for the Fc fragment of the immunoglobulins and it is thus possible to cause the viral RNA associated with the immunoglobulins to penetrate into these permissive cells, via the interaction between the Fc fragment of the immunoglobulins and one of its membrane receptors, and to bring about the propagation and the replication of the HCV virus in vitro.

Accordingly, the subject of the present invention is also a method for the in vitro culture of the HCV virus, according to which the LVP/Ig complex (preferably purified according to the method of the invention) and permissive cells which contain at their surface at least one type of receptor for the Fc fragment of the immunoglobulins or permissive cells expressing at least one receptor for a molecule having the capacity to bind the immunoglobulins are brought into contact, in a given medium and under appropriate conditions, the said permissive cells being capable of allowing the propagation and the replication of the HCV virus in vitro. Of course, the expression of this or these receptors may be either spontaneous or induced, in particular by transfection.

Among the permissive cells expressing at least one type of receptor for the Fc fragment of the immunoglobulins, there may be mentioned by way of nonlimiting examples mononuclear cells (stem cells derived from the bone marrow, monoblasts, promonocytes, monocytes and macrophages) macrophage precursor cells, B lymphocytes, NK cells, hepatocytes, dendritic cells, epithelial cells, vascular endothelium cells, mastocytes, Langerhans' cells; syncytiotrophoblasts, eosinophilic, basophilic and neutrophilic polynuclear cells, platelets.

Among the permissive cells expressing at least one receptor for a molecule having the capacity to bind immunoglobulins, there may be mentioned erythocytes and their precursors, macrophages, monocytes, polymorphonuclear leukocytes (eosinophilic, basophilic and neutrophilic polynuclear cells), B lymphocytes and dendritic cells.

The macrophages are preferably chosen from the group which consists of histiocytes, alveolar macrophages, macrophages of the spleen and of the lymphoid tissue, Kuppfer cells, osteoclasts, type A synovial cells, tissue macrophages and precursor cells from which these cells are derived. Because the HCV virus is hepatotropic, the cells of human or animal primary hepatocytes, the cells of the group of human or animal hepatocarcinoma cell lines and the Kuppfer cells are advantageously preferred. However, since it has been shown that HCV is capable of being propagated and of replicating in the lymphocytes, the B lymphocytes are also preferred permissive cells.

When the permissive cells are cells of human or animal primary hepatocytes, cells of the group of human or animal hepatocarcinoma cell lines or Kuppfer cells, the infection of the said cells by the HCV virus is carried out via the interaction between the Fc fragment of the human immunoglobulins and at least one of the receptors for the Fc fragment present at the surface of the said permissive cells but also via the interaction of the LVPs which are ligands for a route of endocytosis associated with the receptors for the lipoproteins, such as the LDL-receptor and the LSR-receptor which are present at the surface of these same permissive cells.

The term HCV virus refers to any viral species, among which the strains which are pathogenic for humans, the attenuated strains and the defective strains derived from the said strains. Indeed, it is known that the RNA viruses exhibit a high rate of spontaneous mutations. Multiple strains may therefore exist which may be virulent to a greater or lesser degree. It is within the capability of persons skilled in the art to identify such strains, for example by nucleic and/or peptide sequence homology relative to a reference strain and/or by identifying a strain or an isolate with respect to morphological and/or immunological criteria.

The term "in vitro" cellular system refers to cells which have been replicated in vitro and therefore includes the primary cultures, the cultures derived from primary cultures, the primary lines and the lines derived from the said primary lines. Because it is known that induced or spontaneous modifications may occur in the karyotype during storage or transfer, the cells derived from a reference cell line may not be strictly identical to the original cells or cultures and the invention includes such variants.

The term cell line refers to the established, immortalized or spontaneous lines. In practice, to carry out a viral culture of interest, it is necessary for the virus to be able to propagate in vitro in a culture in which the cells are capable of multiplying permanently and thus allow the viral propagation. The cell line is therefore preferably an established cell line or a cell line which results from immortalization by various methods. This may be carried out (i) by the establishment of a stable, established or continuous line or by coculturing permissive cells with tumorized permissive cells of the same nature, which are capable of multiplying indefinitely and of allowing the propagation of the virus within the culture, the viral inoculation taking place within the culture (ii) using primary cells infected with the virus which are then cocultured with permissive tumorized cells which allow the propagation of the virus within the culture of the cell line thus established or (iii) by viral infection of a cell line, for example an immortalized line of B lymphocytes, for example by the Epstein-Barr virus.

The appropriate medium selected for the HCV culture is the RPMI medium or a medium derived from the RPMI medium. Preferably, it is the RPMI 1640 medium supplemented with:

penicillin 1%

streptomycin 1%

glutamine 2 mM

decomplementized FCS (foetal calf serum) 10%.

Optionally, for the culture of certain permissive cells, the abovementioned medium comprises, furthermore, 50 .mu.M beta-mercaptoethanol.

Several passages of permissive cells thus infected are carried out and the presence of the said virus is detected in the infected permissive cells and in the culture supernatant by RT-PCR and/or by an immunological technique, such as by indirect immunofluorescence using an antibody specific for the said virus and/or by flow cytometry.

The method of the invention, which makes it possible to obtain propagation of the HCV virus, is useful in particular for studying its replication mechanism, for testing neutralizing antibodies and antiviral agents, and for developing biological materials for diagnosis and therapy. Moreover, the method of the invention makes it possible to obtain an infected cell line useful for screening and/or selecting at least one antiviral molecule, by bringing the infected cell line and the antiviral molecule into contact.

The invention also relates to a method for preparing a composition for the detection, in a sample, of antibodies directed against HCV which comprises at least a partial or complete purification of the viral particles of the said virus or of the polypeptides obtained from the method of the invention. The expression partial or complete purification is understood to mean, for example, a purification by ultracentrifugation on a sucrose gradient, by differential precipitation in ammonium sulphate, a gel chromatography or any other method well known to persons skilled in the art. In particular, the said viral particles or the said polypeptides are attached to a solid support.

Moreover, the invention relates to a method for producing antibodies or fragments of antibodies directed against the HCV virus according to which an animal is immunized with the complex consisting of LVPs associated with human immunoglobulins having a density of less than 1.063 g/ml, preferably of between 1.0063 and 1.063 g/ml or with fractions of the said complex; the said complex being optionally prepared according to the method described above. The complex may thus be subjected to a step of treatment prior to the immunization, for example by treating with detergents or chaotropic agents, for the production of fractions consisting of viral proteins, lipids and phospholipids. The production of polyclonal and monoclonal antibodies or fragments of antibodies forms part of the general knowledge of persons skilled in the art. There may be mentioned, by way of example, Kohler G. and Milstein C. (1975) Continuous culture of fused cells secreting antibody of predefined specificity, Nature, 256: 495-497 and Galfre G. et al. (1977) Nature, 266: 522-550 for the production of polyclonal antibodies and Roda A., Bolelli G. F. Production of high titer antibody to bile acids, Journal os Steroid Biochemistry, Vol. 13, pp 449-454 (1980) for the production of polyclonal antibodies. The antibodies are produced by immunizing mice or rabbits with the LVP/Ig+ complex or with fractions of the said complex comprising viral proteins, lipids and phospholipids. The animals are subjected to an injection of immunogen using complete Freund's adjuvant. The sera and the hybridome culture supernatants obtained from the immunized animals are analyzed for their specificity and their selectivity using conventional techniques, such as for example ELISA or Western blot tests. The hybridomes producing the most specific and the most sensitive antibodies are selected. Monoclonal antibodies may also be produced in vitro by cell culture of the hybridomes produced or by recovering ascitic fluid, after intraperitoneal injection of the hybridomes into mice. Regardless of the mode of production, as supernatant or as ascites, the antibodies are then purified. The methods of purification used are essentially filtration on ion-exchange gel and exclusion chromatography or immunoprecipitation. A sufficient number of antibodies are screened in function tests to identify the most efficient antibodies. The in vitro production of antibodies, of fragments of antibodies or of derivatives of antibodies, such as chimeric antibodies produced by genetic engineering is well known to persons skilled in the art.

More particularly, the expression fragment of antibodies is understood to mean the F(ab)2, Fab, Fab', sFv (Blazar et al., 1997, Journal of Immunology 159: 5821-5833 and Bird et al., 1988, Science 242: 423-426) fragments of a native antibody and the expression derivative is understood to mean, inter alia, a chimeric derivative of a native antibody (see for example Arakawa et al., 1996, J. Biochem 120: 657-662 and Chaudray et al., 1989, Nature 339: 394-397).

The invention also relates to a method for the in vitro culture of the HCV virus according to which a complex consisting of LVPs associated with human immunoglobulins (LVP/Ig) as defined above is made available, the said complex is brought into contact with protein A for the production of an LVP/Ig/protein A complex, the said LVP/Ig/protein A complex is brought into contact with at least one antibody specific for at least one cell receptor for the production of an LVP/Ig/protein A/antibody complex and the said LVP/Ig/protein A/antibody complex and permissive cells which contain or express at their surface at least one cell receptor having the capacity to bind the antibody are brought into contact, in a culture medium and under appropriate conditions; the said permissive cells being capable of allowing the propagation and the replication of the HCV virus in vitro. According to this method and preferably:

protein A is coupled to a support, such as beads or sepharose,

the antibody is antibody specific for at least one of the receptors for the LDLs and the permissive cells contain or express at their surface at least one receptor for the LDLs,

the permissive cells are chosen from human or animal primary hepatocytes and the cells of the group of human or animal hepatocarcinoma cell lines, such as the cells of the human heptacarcinoma line HepG2,

the culture medium is the DMEM medium supplemented with 10% FCS,

the presence of the HCV virus in the permissive cells is detected by RT-PCR and/or by immunological technique, such as by indirect immunofluorescence, in particular using an antibody specific for the said virus and/or by flow cytometry.

In the method of the invention, protein A is added in excess, which means that the protein A which is not bound to the human immunoglobulins of the LVP/Ig is still available, adsorbed to the surface of the LVPs, to bind to the antibody specific for a given cell receptor. Of course, the antibody may be a polyclonal or monoclonal antibody, but preferably will be a monoclonal antibody to ensure better specificity.

The invention also relates to a method for preparing a composition for the detection, in a sample, of antibodies directed against the HCV virus which comprises at least a partial or complete purification of the HCV viral particles or of the polypeptides obtained from a method of culture as defined above. Preferably for the preparation of the said composition, the viral particles or the polypeptides are attached to a solid support.

The subject of the invention is also a method for charging in vitro antigen presenting cells (APCs), according to which a complex consisting of LVPs associated with human immunoglobulins (LVP/Ig) as defined in either of claims 1 and 2 is made available, the said complex is brought into contact with protein A for the production of an LVP/Ig/protein A complex, the said LVP/Ig/protein A complex is brought into contact with at least one antibody specific for at least one cell receptor of the antigen presenting cells (APCs) collected from a human being or an animal for the production of an LVP/Ig/protein A/antibody complex and the said LVP/Ig/protein A/specific antibody complex is brought into contact with the said antigen presenting cells. The APCs are involved in the process of preparing the exogenous antigens to produce immunogenic peptides which bind to the molecules of the Major Histocompatibility Complex class I or II allowing the stimulation of the lymphocytes. The term APCs generally covers the macrophages, the B cells and the dendritic cells. In the present invention, there is particular focus on the dendritic cells, but this is not at all limiting and the method covers any antibody which is capable of binding to at least one receptor of the APCs. When the antigen presenting cells are dendritic cells, the antibody is an antibody specific for at least one cell receptor of the dendritic cells which is chosen from the "scavenger" receptors A and B, the mannose receptor and the "Toll Like Receptors" (TLRs). In the method of charging of the invention, the APCs are autologous, which means that they are collected from a human being or an animal and that after charging in vitro according to the method of the invention they are reintroduced into the same human being or animal to induce a humoral and cellular response. Reference is generally made to cell therapy.

The invention also relates to the charged APCs which can be obtained according to the method described above and to a therapeutic composition intended to induce a humoral and cellular response in a human being or an animal comprising a therapeutic agent which consists of antigen presenting cells charged according to the method of the invention. Furthermore, the invention also covers the antigen presenting vesicles or "exosomes" which are capable of being obtained from the APCs. The said composition comprises the usual adjuvants and/or diluents and/or excipients for the preparation of a therapeutic or prophylactic composition for the treatment or prophylaxis of immunological disorders or infections, provided that they are pharmaceutically acceptable.

The definitions of the pharmaceutically acceptable excipients, diluents and adjuvants are given in Remington's Pharmaceutical Sciences 16th ed., Mack Publishing Co.

Thus according to the invention, the method for the treatment or prophylaxis of an HCV virus infection consists in administering to a human being or to an animal, at predetermined doses, a therapeutic composition as defined above.

The invention also relates to a method for the production of antibodies or fragments of antibodies directed against the HCV virus, according to which an animal is immunized with an LVP/Ig/protein A/antibody complex which can be obtained by a method according to which, a complex consisting of LVPs associated with human immunoglobulins (LVP/Ig) as defined above is made available, the said complex is brought into contact with protein A for the production of an LVP/Ig/protein A complex, the said LVP/Ig/protein A complex is brought into contact with at least one antibody specific for at least one cell receptor of antigen presenting cells (APCs) for the production of an LVP/Ig/protein A/antibody complex. Preferably, the antigen presenting cells are dendritic cells and the antibody is an antibody specific for at least one cell receptor of the dendritic cells chosen from the "scavenger" receptors A and B, the mannose receptor and the "Toll Like Receptors" (TLRs)

Claim 1 of 27 Claims

What is claimed is:

1. Complex consisting of lipo-viro particles (LVPs) associated with human immunoglobulins having a density of less than 1.063 g/ml.
 

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