Title:
ANTIBODIES DIRECTED AGAINST HEPATITIS C VIRUS E1E2 COMPLEX, COMPOSITIONS OF HCV PARTICLES, AND PHARMACEUTICAL COMPOSITIONS
Kind Code:
A1


Abstract:
The invention provides new conformational antibodies, and more particular conformational monoclonal antibodies and fragments thereof, that are directed against HCV. Also provided are compositions of particles that are recognized by such antibodies, and pharmaceutical compositions containing these particles. Also described are HCV enveloped subviral particles and purified HCV enveloped complete viral particles, as well as processes for their preparation.



Inventors:
Petit, Marie Anne (LYON CEDEX 08, FR)
Drouet, Emmanuel (Grenoble cedex 9, FR)
Trepo, Christian (LYON CEDEX 08, FR)
Application Number:
13/114229
Publication Date:
12/22/2011
Filing Date:
05/24/2011
Assignee:
PETIT MARIE ANNE
DROUET EMMANUEL
TREPO CHRISTIAN
Primary Class:
Other Classes:
435/325, 435/358, 435/365, 530/387.3, 530/388.3, 530/389.4, 536/23.53, 435/243
International Classes:
A61K39/395; A61K39/29; A61K39/42; A61P31/14; C07K14/18; C07K16/00; C07K16/10; C07K16/46; C12N1/00; C12N5/10; C12N5/20; C12N7/00; C12N7/02; C12N7/04; C12N15/13; C12N15/63; G01N33/576
View Patent Images:



Primary Examiner:
BOESEN, AGNIESZKA
Attorney, Agent or Firm:
YOUNG & THOMPSON (209 Madison Street Suite 500, Alexandria, VA, 22314, US)
Claims:
What is claimed is:

1. An isolated antibody, or a functional fragment thereof, comprising: (a) at least one heavy chain, or fragment thereof, having an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 34, or a conservative variant thereof, or an antigen-binding portion thereof; or (b) at least one light chain, or fragment thereof, having an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 46, or a conservative variant thereof, or an antigen-binding portion thereof; or (c) at least one heavy chain, or fragment thereof, having an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 34, a conservative variant thereof, or an antigen-binding portion thereof, and at least one light chain, or fragment thereof, having an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 46, or a conservative variant thereof, or an antigen-binding portion thereof.

2. The isolated antibody, or a functional fragment thereof, according to claim 1, wherein: the antigen-binding portion of SEQ ID NO: 34 is a heavy chain variable region having an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 35, or a conservative variant thereof; and the antigen-binding portion of SEQ ID NO: 46 is a light chain variable region having an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 47, or a conservative variant thereof.

3. The isolated antibody, or a functional fragment thereof, according to claim 1, wherein: the antigen-binding portion of SEQ ID NO: 34 comprises three complementary determining regions having amino acid sequences set forth in SEQ ID NO: 43, SEQ ID NO: 44 and SEQ ID NO: 45 or conservative variants thereof; and the antigen-binding portion of SEQ ID NO: 46 comprises three complementary determining regions having amino acid sequences set forth in in SEQ ID NO: 51, SEQ ID NO: 52 and SEQ ID NO: 53 or conservative variants thereof.

4. The isolated antibody, or a functional fragment thereof, according to claim 1, wherein the antibody is a chimeric, humanized or deimmunized monoclonal antibody.

5. An antigen-binding molecule comprising: (a) three complementary determining regions having amino acid sequences set forth in SEQ ID NO: 43, SEQ ID NO: 44 and SEQ ID NO: 45 or conservative variants thereof; or (b) three complementary determining regions having amino acid sequences set forth in in SEQ ID NO: 51, SEQ ID NO: 52 and SEQ ID NO: 53 or conservative variants thereof; or (c) three complementary determining regions having amino acid sequences set forth in SEQ ID NO: 43, SEQ ID NO: 44 and SEQ ID NO: 45 or conservative variants thereof, and three complementary determining regions having amino acid sequences set forth in in SEQ ID NO: 51, SEQ ID NO: 52 and SEQ ID NO: 53 or conservative variants thereof.

6. A pharmaceutical composition comprising at least one isolated antibody, or functional fragment thereof, according to claim 1 and a pharmaceutically acceptable carrier or excipient.

7. An isolated nucleic acid molecule encoding an antibody, or functional fragment thereof, according to claim 1, comprising: (a) a polynucleotide having a sequence selected from the group consisting of the sequence set forth in SEQ ID NO: 54, homologous variants thereof, and fragments thereof that encode antigen-binding portions of the antibody heavy chain; or (b) a polypeptide having a sequence selected from the group consisting of the sequence set forth in SEQ ID NO: 55, homologous variants thereof, and fragments thereof that encode antigen-binding portions of the antibody light chain; or (c) a first polypeptide having a sequence selected from the group consisting of the sequence set forth in SEQ ID NO: 54, homologous variants thereof, and fragments thereof that encode antigen-binding portions of the antibody heavy chain, and a second polypeptide having a sequence selected from the group consisting of the sequence set forth in SEQ ID NO: 55, homologous variants thereof, and fragments thereof that encode antigen-binding portions of the antibody light chain.

8. An isolated expression vector comprising the isolated nucleic acid molecule according to claim 7.

9. An isolated host cell comprising the isolated expression vector according to claim 8.

10. The isolated host cell according to claim 9, wherein the host cell is prokaryotic or eukaryotic.

11. A pharmaceutical composition comprising at least one isolated antibody, or functional fragment thereof, an antigen-binding molecule according to claim 5 and a pharmaceutically acceptable carrier or excipient.

Description:

RELATED APPLICATIONS

This application is a continuation-in-part of currently pending U.S. application Ser. No. 12/408,080, filed on Mar. 20, 2009, which is a divisional application of U.S. application Ser. No. 10/550,295, filed on Feb. 23, 2006, now U.S. Pat. No. 7,524,650, which is the 35 U.S.C. 371 national stage of International application PCT/EP04/03412 filed on Mar. 31, 2004, and which claims priority to European Application No. 03 290 822.0, filed on Apr. 1, 2003. The teachings and the entire content of each of the above-identified applications are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

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: a 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 reasons why antibodies that 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 the 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.

BRIEF SUMMARY OF THE INVENTION

Thus, in one aspect, the present invention provides antibodies that react with natural HCV particles.

In particular, the present invention provides an isolated antibody, or functional fragment thereof, comprising: (a) at least one heavy chain, or fragment thereof, having an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 34, or a conservative variant thereof, or an antigen-binding portion thereof; or (b) at least one light chain, or fragment thereof, having an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 46, or a conservative variant thereof, or an antigen-binding portion thereo; or (c) at least one heavy chain, or fragment thereof, having an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 34, a conservative variant thereof, or an antigen-binding portion thereof, and at least one light chain, or fragment thereof, having an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 46, or a conservative variant thereof, or an antigen-binding portion thereof.

In certain embodiments, the antigen-binding portion of SEQ ID NO: 34 is a heavy chain variable region having an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 35, or a conservative variant thereof. In other embodiments, the antigen-binding portion of SEQ ID NO: 34 comprises three complementary determining regions having amino acid sequences set forth in SEQ ID NO: 43, SEQ ID NO: 44 and SEQ ID NO: 45 or conservative variants thereof.

In certain embodiments, the antigen-binding portion of SEQ ID NO: 46 is a light chain variable region having an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 47, or a conservative variant thereof. In other embodiments, the antigen-binding portion of SEQ ID NO: 46 comprises three complementary determining regions having amino acid sequences set forth in in SEQ ID NO: 51, SEQ ID NO: 52 and SEQ ID NO: 53 or conservative variants thereof.

In certain embodiment, the isolated antibody is a chimeric, humanized or deimmunized monoclonal antibody.

In another aspect, the present invention provides an antigen-binding molecule comprising: (a) three complementary determining regions having amino acid sequences set forth in SEQ ID NO: 43, SEQ ID NO: 44 and SEQ ID NO: 45 or conservative variants thereof; or (b) three complementary determining regions having amino acid sequences set forth in in SEQ ID NO: 51, SEQ ID NO: 52 and SEQ ID NO: 53 or conservative variants thereof; or (c) three complementary determining regions having amino acid sequences set forth in SEQ ID NO: 43, SEQ ID NO: 44 and SEQ ID NO: 45 or conservative variants thereof, and three complementary determining regions having amino acid sequences set forth in in SEQ ID NO: 51, SEQ ID NO: 52 and SEQ ID NO: 53 or conservative variants thereof.

In yet another aspect, the present invention provides a pharmaceutical composition comprising at least one isolated antibody, or functional fragment thereof, as described herein and a pharmaceutically acceptable carrier or excipient.

The present invention also provides a pharmaceutical composition comprising at least one antigen-binding molecule as described herein and a pharmaceutically acceptable carrier or excipient.

In another aspect, the present invention provides an isolated nucleic acid molecule encoding an antibody, or functional fragment thereof, as described herein, wherein the nucleic acid molecule comprises: (a) a polynucleotide having a sequence selected from the group consisting of the sequence set forth in SEQ ID NO: 54, homologous variants thereof, and fragments thereof that encode antigen-binding portions of the antibody heavy chain; or (b) a polypeptide having a sequence selected from the group consisting of the sequence set forth in SEQ ID NO: 55, homologous variants thereof, and fragments thereof that encode antigen-binding portions of the antibody light chain; or (c) a first polypeptide having a sequence selected from the group consisting of the sequence set forth in SEQ ID NO: 54, homologous variants thereof, and fragments thereof that encode antigen-binding portions of the antibody heavy chain, and a second polypeptide having a sequence selected from the group consisting of the sequence set forth in SEQ ID NO: 55, homologous variants thereof, and fragments thereof that encode antigen-binding portions of the antibody light chain.

In another aspect, the present invention provides an isolated expression vector comprising an isolated nucleic acid according to the invention.

In another aspect, the present invention provides an isolated host cell comprising an isolated expression vector of the invention.

In certain embodiments, the host cell is a prokaryotic cell. In other embodiments, the host cell is a eukaryotic cell.

In still another aspect, the invention provides compositions of natural HCV particles, in sufficient quantity and concentration to allow sufficient immunization of antibody producing animals.

In yet another aspect, the invention provides specific HCV compositions devoid of infectivity, liable to be used as active substances of pharmaceutical compositions.

These and other objects, advantages and features of the present invention will become apparent to those of ordinary skill in the art having read the following detailed description of the preferred embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a set of sodium dodecyl sulfate-polyacrylamid gel electrophoresis (SDS-PAGE) of antibody precipitated lysates of 35S labelled cells. In each gel, lanes 1, 2, 3, 4, 5, 6 and 7 correspond to antibodies C9.19.16, C2.22.1, D32.10, D3.20.12, C7.24.19, C7.14.41 and C1.9.3, respectively, and MW corresponds to a molecular weight marker. The sizes of the bands corresponding to the molecular weight marker, are presented on the right of the gel. When possible the bands are identified on the left of the gel as E1, E2 or Agg (for aggregated). SDS-PAGE was obtained for antibody precipitated lysates of 35S labelled control cells under reducing conditions (A); of 35S labelled cells expressing HCV E1 protein under reducing conditions (B); of 35S labelled cells expressing HCV E2 protein under reducing conditions (C); of 35S labelled cells expressing HCV E1E2 protein under reducing conditions (D); and of 35S labelled cells expressing HCV E1E2 protein under non-reducing conditions (E).

FIG. 2 presents the results of a Western blot of HCV viral particles using the D32.10 monoclonal antibody under non-reducing and reducing conditions. In the first Western blot, M represents a molecular weight marker under non-reducing conditions, the size of the bands of the marker are indicated on the left of the gel (in kDa); and lane 1 represents HCV viral particles using the D32.10 monoclonal antibody under non-reducing conditions. In the second Western blot, M represents a molecular weight marker under reducing conditions; Lane 2 represents 2.5 rig of HCV viral particles using the D32.10 monoclonal antibody under reducing conditions; and Lane 3 represents 5 μg of HCV viral particles using the D32.10 monoclonal antibody, under reducing conditions. The size of several bands of Lane 3 is indicated (in kDa) on the right side of the gel, some correspond to HCV protein E2 (60 and 68), others to HCV protein E1 (34 and 31).

FIG. 3 presents the results of a Western blot of HCV viral particles submitted to deglycosylation by glycosidase A and endoglycosidase H (B). In (A), lane M represents a molecular weight marker, with the size of three bands indicated (in kDa) on the left of the gel; lanes 1, 2, 3 and 4 represent glycosidase at a concentration of 20, 10, 5 and 0 mU/ml, respectively. On the right of the gel, the positions of HCV proteins E2 and E1 are indicated, as well as the position of a major deglycosylated form of protein E1 (E1*), the size (in kDa) of several bands corresponding to deglycosylated forms of E1 or E2, and the major deglycosylated form of E2 (41*). In (B), lane M represents a molecular weight marker, with the size of four bands indicated (in kDa) on the left of the gel; lanes 1 and 2 represent a control deglycosylation experiment carried out without endoglycosidase H and a deglycosylation experiment carried out in presence of endoglycosidase H, respectively. On the right of the gel are indicated the major bands corresponding to the fully glycosylated forms of E2 and E1, and to the deglycosylated forms of E2 (50, 48, 46 and 42 kDa) and E1 (28 and 24 kDA). The predominant deglycosylated forms of E2 (50*) and E1 (28*) are indicated by a star.

FIG. 4 is a graph, which represents the characterization of the fractions obtained by centrifugation on a sucrose density gradient of a HCV viral particles preparation. Three parameters have been measured: HCV RNA content, HCV core protein content and reactivity towards the D32.10 monoclonal antibody. The horizontal axis represents the numbers of the fractions submitted to characterization. The left vertical axis represents HCV RNA concentration (×105 UI/ml) and a measure of the reactivity towards D32.10 as determined by indirect EIA (OD at 450 nm). The right vertical axis represents the HCV core protein concentration (in pg/ml). The curve with the black dots represents the reactivity of the different fractions towards D32.10, the curve with the white triangles represents the fractions contents in core protein and the dotted bars represent the fractions contents in HCV RNA.

FIG. 5 is a set of transmission electron microscopy pictures of a HCV viral particles preparation (A), a HCV enveloped complete viral particles preparation (B) and a HCV enveloped subviral particles preparation (C). In (A), the horizontal bar represents a scale of 200 nm in length. The larger circular elements are HCV enveloped complete viral particles and the smaller circular elements are HCV enveloped subviral particles. In (B), the horizontal bar represents a scale of 50 nm in length, and the circular elements are HCV enveloped complete viral particles. In (C), the horizontal bar represents a scale of 50 nm in length, and the circular elements are HCV enveloped subviral particles.

FIG. 6 presents the results of a Western blot of HCV viral particles using the D4.12.9 monoclonal antibody. Each Lane M represents a molecular weight marker, with the size of four bands (75, 50, 37, 25) indicated (in kDa) on the left side and on the right side of the gel. Lanes 1 and 2 represent the result of a digestion of the HCV viral particles by glycosidase A and by endoglycosidase H, respectively. Lanes 3 and 4 represent the result of a proteolytic digestion of HCV viral particles by proteases trypsin and V8, lane 5 represents the result of lysis by NP-40, and in lane 6 no prior treatment as been performed. The two major forms of undigested E2 protein (68 and 45) are indicated (in kDa) on the right side of the gel, and the major deglycosylated form of E2 (E2*) is indicated on the left side of the gel.

FIG. 7 is a graph presenting a D4.12.9 based characterization of fractions obtained by centrifugation on a sucrose density gradient of a HCV viral particles preparation. In addition to the fractions reactivity towards D4.12.9, the fractions contents in core protein was also measured. The horizontal axis represents the numbers of the fractions submitted to characterization. The right vertical axis represents a measure of the reactivity towards D4.12.9 as determined by indirect EIA (OD at 450 nm), and the left vertical axis, the HCV core protein concentration (in pg/ml). The curve with the black squares shows the reactivity of the different fractions towards D4.12.9, the curve with the black diamonds shows the fractions contents in core protein.

FIG. 8 is a set of three graphs showing the immunoreactivity of three biotinylated peptides comprising amino acids 290-306 (A), amino acids 480-494 (B) and amino acids 608-622 (C), respectively towards the serum of 55 healthy individuals (11 sera, numbered T1 to T11 on the horizontal axis) and of HCV infected patients (44 sera, numbered A1 to A44 on the horizontal axis). The vertical axis represents the reactivity of the sera to the three peptides as measured by ELISA (OD at 492 nm×1000). The white bars represent the reactivity of the serum of healthy individuals and the grey bars represent the reactivity of the serum of infected patients. The horizontal line represents a cut-off value above which a serum response is considered positive. In (A), the cut-off value is 0.691, in (B), 0.572 and in (C), 0.321.

FIG. 9 shows the results of a quantification of HCV core protein using the Total HCV core antigen assay from Ortho Clinical Diagnostics (A) and analysis by Western blotting (B) in HCV-Fan pellet and the fractions from 30 to 45% sucrose gradient of type 1 described above. In (A), all the fractions were tested at a dilution of 1:2 in THE buffer. HCV-Fan pellet was tested at dilutions 1:25, 1:50 and 1:100, and found to contain 332 pg/ml. The cutoff value was 1.4 pg/ml. In (B), HCV-Fan pellet and fractions 4, 8, 12 and 13 from the 30-45% sucrose gradient were subjected to SDS-12.5% PAGE. HCV core proteins were detected using a mixture of monoclonal antibodies anti-core, 7G12A8, 2G9A7 and 19D9D6 (5 μ/ml each). Numbers on the left indicate molecular weights of markers (M) (in kDa), and the HCV core proteins are indicated on the right.

FIG. 10 is a graph showing the results of a quantitative RT-PCR analysis of HCV RNA was analyzed by the quantitative RT-PCR using the Amplicor HCV Monitor version 2.0 test (Roche Diagnostics) in all the fractions from the initial 10 to 60% sucrose gradient. The results were calculated using the QS International Units (IU) per PCR, which was specific for each lot, and expressed in IU HCV RNA/ml.

FIG. 11 shows the results of an analysis of serum-derived HCV enveloped particles by electron microscopy (EM). A HCV-Fan pellet (2 μg) was immunoprecipitated using MAb D32.10 (5 μg) (panels a, b, c and d) or irrelevant MAb (panel e), and, after ultracentrifugation, the immune-complexes were absorbed onto grids and stained with 1% uranyl acetate (pH 4.5). (A) The particles were found to vary somewhat in diameter (expressed in nm), and a histogram was performed on 156 viral particles (VPs), showing a particle size distribution with a peak centered at 35 nm (A). Electron micrographs are shown in (B) where the horizontal bars in panels a, b, c, and e indicate 50 nm

FIG. 12 is a set of two graphs showing the results of an electron microscopy analysis of HCV-Fan particles from sucrose gradient peaks 1 (A) and 2 (B) after immunoprecipitation using D32.10, adsorption on grids and staining. The graph in (A) is a histogram performed on 41 viral particles, showing that the predominant species (85.4%) in peak 1 is a spherical particle with a diameter of about 20 nm, called <<small particles>>. The graph in (B) is a histogram performed on 89 viral particles, showing that the most prevalent forms (77.5%) in peak 2 has a mean diameter of about 41 nm Peak 2 appeared somewhat more heterogeneous in size, and consisted primarily of 35 and 50-nm-diameter particles, called <<large particles>>. The bars represent 50 nm

FIG. 13 shows pictures of indirectly immuno-gold labelled of enveloped HCV particles. Microscope grids coated with the D32-10-immune complexes (HCV pellet, 20 n; MAb D32.10, 5 μg) were incubated with a 1:50 dilution of goat anti-mouse IgG-conjugated colloidal gold particles (10 nm), as second antibody. According to steric hindrance of IgG (about 15 nm in EM), only few gold-particles could identify antibody binding. No gold-particles were observed outside the viral particles. Bars in all panels indicate 20 nm.

FIG. 14(A)-(B) shows results of an isopycnic centrifugation in sucrose density gradient (10% to 60%) of HCV-enriched pellets from the isolate HCV-L of genotype 1b. (A) is a graph showing the density (left horizontal axis, g/ml) and the E1E2-D32.10 reactivity (right vertical axis, A490 nm) of the different fractions (horizontal axis) obtained after centrifugation. (B) shows the results of a Western blot of fractions 6, 8, 10, 12 and 14, using the anti-E1E2 D32.10 MAb. The numbers on the left indicate molecular weights of markers (M) (in kDa), and the HCV E1 and E2 proteins were are indicated on the right. FIG. 14(C)-(D) shows results of the isopycnic centrifugation in sucrose density gradient (10% to 60%) of HCV-enriched pellets from the isolate HCV-Fan of genotype 1a/2a. (C) is a graph showing the density (left horizontal axis, g/ml) and the E1E2-D32.10 reactivity (right vertical axis, A490 nm) of the different fractions (horizontal axis) obtained after centrifugation. (D) shows the results of a Western blot of fractions 6, 8, 10, 12 and 14 using the anti-E1E2 D32.10 MAb. For an EIA analysis, each fraction was diluted at 1/10000 (A and C), and for Western blotting 5 μl upper lane of the selected fractions was used (B and D). The density of the fractions was determined by refractometry and expressed in g/ml. Absorbance (A) was determined at 490 nm. The results of EIA were considered as positive when higher than a cut-off value, corresponding to the mean of at least three negative controls multiplied by 2.1.

FIG. 15 is a set of three graphs showing the results of isopycnic centrifugations of an HCV-Fan pellet. In (A) the centrifugation was performed using a 30 to 45% sucrose gradient of type 1 (2 ml of 30%, 3 ml of 35%, 3 ml of 40%, 2 ml of 45%). Particles from sucrose gradient (30-45%) peaks 1 (fraction 8) and 2 (fractions 12 and 13) were then subjected individually to a second equilibrium centrifugation (B and C, respectively). Peak 1 was centrifuged in a 30 to 45% sucrose gradient of type 2 (3 ml of 30%, 3 ml of 35%, 2 ml of 40%, 2 ml of 45%) (B). Peak 2 was centrifuged in a 30 to 45% sucrose gradient of type 3 (2 ml of 30%, 2 ml of 35%, 3 ml of 40%, 3 ml of 45%) (C). The density of fractions determined by refractometry is expressed in g/ml. E1E2-D32.10 reactivity was analyzed by indirect EIA.

DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS

I—Antibodies and Fragments Thereof

In certain embodiments, the present invention relates to conformational antibodies that are capable of specifically binding to the natural HCV viral envelope. The term “conformational antibody” designates an antibody which recognizes an epitope having a three-dimensional structure defined by its molecular surrounding. The expression “specifically binding” means that the antibody binds to an epitope which is found on substantially only one of the elements forming the natural HCV viral envelope, i.e., the antibody exhibits substantially no binding with elements other than the elements forming the natural HCV envelope. The binding specificity of an antibody can, for example, be tested using methods well known to the man skilled in the art, such as Western blotting, where a biological sample is electrophoretically migrated on a gel, then transferred onto a membrane and co-incubated with the antibody to be tested, which is then detected by a secondary antibody. An antibody is said to be “specifically binding” to a target compound contained in a biological sample if substantially all of the electrophoretical bands detected contain the target compound or parts thereof.

The term “HCV” means “hepatitis C virus”, as described for example in Choo et al. (1989, 1991). HCV comprises in particular RNA, a capsid made of a core protein, and an envelope which comprises lipids and proteins, particularly glycoproteins. The “HCV viral envelope” is made of lipids and proteins, in particular glycoproteins such as HCV proteins E1 and E2 (Clarke, 1997). As used herein, the term “natural HCV” means that HCV, or parts thereof, are as found in biological samples and possibly as isolated and if needed as purified from biological samples. Such samples may be blood, plasma or sera of patients infected by HCV. In particular, “natural” refers to HCV, or parts thereof, which have not been produced by recombinant methods, or by using cell lines or animals, and are different from HCV elements described in Schalich et al. (1996), Blanchard et al. (2002) or WO 92/07001 for instance.

In certain embodiments, the present invention more particularly relates to conformational antibodies that are capable of specifically binding to the natural HCV E2 protein. HCV E2 is described, for example, in Dubuisson (2000) and Op De Beeck et al. (2001). It is produced as a polyprotein (3012 amino acids) which is cleaved to yield E2 (amino acids 384 to 714). As used herein, the term “specifically binding” means that the antibody binds to an epitope, or a part thereof, which is found substantially only on the HCV E2 protein. In particular the antibody can not be detached from the HCV E2 protein in competition tests, where the antibody-E2 protein complex is put in the presence of other proteins. The expression “natural HCV E2 protein”, as used herein, means that the E2 protein is as found in biological samples of patients infected by HCV, in particular the natural HCV E2 protein is not a recombinant protein.

In certain embodiments, the present invention relates to conformational antibodies as defined above, that are capable of neutralizing HCV infections in patients. As used herein, the term “neutralizing HCV infection” means that the antibody is capable of improving the health of patients infected by HCV, as can be evidenced, for instance, by the decrease in HCV detected in blood, plasma or sera. Alternatively or additionally, “neutralizing HCV infection” means that the antibody is capable of preventing individuals to be infected by HCV. The HCV infection neutralization capability of an antibody can be monitored, for example, in model animals, such as chimpanzees or mice, in particular humanized mice, which are chronically infected by HCV, or which are primo-infected by HCV in the presence of said antibody. The antibody tested should be capable of inducing a decrease in HCV related viremia or preventing infection by HCV, respectively.

In certain embodiments, the present invention relates to conformational antibodies as defined above, that are capable of precipitating the HCV E1E2 complex under its covalent form or its non-covalent form. When the HCV E1E2 complex is under a non-covalent form, E1 and E2 are associated by means of weak bonds, such as hydrogen bonds, ionic bonds, Van Der Waals bonds or hydrophobic bonds. When the HCV E1E2 complex is under a covalent form, E1 and E2 are associated by means of covalent bonds, such as disulfure bonds for instance. Covalent and non-covalent forms of the E1E2 complex are described or suggested for example in Deleersnyder et al. (1997). As used herein, the term “precipitating the HCV E1E2 complex” means that the antibody may render the HCV E1E2 complex insoluble. Precipitation may occur for instance such as described by Dubuisson and Rice (1996).

In certain embodiments, the present invention relates to conformational antibodies as defined above, that are capable of specifically binding to the natural HCV E1 protein. HCV E1 is described, for example, in Dubuisson (2000) and Op De Beeck et al. (2001). It is produced as a poly-protein (3012 amino acids) which is cleaved to yield E1 (amino acids 192 to 383). As used herein, the term “specifically binding” means that the antibody binds to an epitope, or a part thereof, which is found substantially only on the HCV E1 protein. In particular the antibody can not be detached from the HCV E1 protein in competition tests, where the antibody-E1 protein complex is put in the presence of other proteins. The expression “natural HCV E1 protein”, as used herein, means that the E1 protein is as found in biological samples of patients infected by HCV, in particular the natural HCV E1 protein is not a recombinant protein.

In certain embodiments, the present invention more specifically relates to conformational antibodies that are capable of specifically binding to the natural HCV E1 protein, to the natural HCV E2 protein, and of precipitating the HCV E1E2 complex under its covalent form or non covalent form.

The present invention relates to a conformational antibody as defined above, that is capable of specifically binding to an epitope constituted of at least one of the following sequences: amino acids 297 to 306 of HCV protein E1; amino acids 480 to 494 of HCV protein E2; and amino acids 613 to 621 of HCV protein E2. Thus, in certain embodiments, an antibody according to the invention is able to bind to: a molecule presenting a peptide comprising amino acids 297 to 306 of HCV protein E1, corresponding to the following sequence: RHWTTQGCNC (SEQ ID NO: 1); and/or to a molecule presenting a peptide comprising amino acids 480 to 494 of HCV protein E2, corresponding to the following sequence: PDQRPYCWHYPPKPC (SEQ ID NO: 2); and/or to a molecule presenting a peptide comprising amino acids 613 to 621 of HCV protein E2, corresponding to the following sequence: YRLWHYPCT (SEQ ID NO: 3).

The binding of such an antibody to at least one of the above-mentioned sequences can be tested by synthesising a peptide containing any of these sequences and by assessing the binding ability of the antibody to the synthesised peptide using methods well known to one skilled in the art, such as ELISA (Enzyme Linked Immuno Sorbent Assay) or EIA (Enzyme Immunoassay) for example.

In certain embodiments, the present invention relates to a conformational antibody as defined above, that is capable of specifically binding to an epitope constituted of each of the following sequences: amino acids 297 to 306 of HCV protein E1; amino acids 480 to 494 of HCV protein E2; and amino acids 613 to 621 of HCV protein E2. An antibody according to such embodiments of the invention is able to bind to a molecule presenting an epitope, said epitope comprising: a peptide comprising amino acids 297 to 306 of HCV protein E1, corresponding to the following sequence: RHWTTQGCNC (SEQ ID NO: 1); and a peptide comprising amino acids 480 to 494 of HCV protein E2, corresponding to the following sequence: PDQRPYCWHYPPKPC (SEQ ID NO: 2); and a peptide comprising amino acids 613 to 621 of HCV protein E2, corresponding to the following sequence: YRLWHYPCT (SEQ ID NO: 3).

The binding of such an antibody to an epitope comprising each of the above-mentioned sequences can be tested by assessing the binding ability of the antibody to a molecule, in particular a protein, which comprises the sequence RHWTTQGCNC (SEQ ID NO: 1), and to a molecule, in particular a protein, which comprises the sequence PDQRPYCWHYPPKPC (SEQ ID NO: 2), and to a molecule, in particular a protein, which comprises the sequence YRLWHYPCT (SEQ ID NO: 3), using methods well known to those skilled in the art, such as ELISA or EIA for example.

In certain embodiments, a conformational antibody as defined above is a monoclonal antibody. The expression “monoclonal antibody” means that the antibody binds to substantially only one epitope, or to parts of said only one epitope. A monoclonal antibody can be obtained from monoclonal cell lines, derived from immortalized antibody secreting cells, such as hybridomas for instance. Monoclonal cell lines are derived from culturing a unique cell. A hybridoma can be produced by fusion of an antibody secreting cell, such as a B cell, with an immortalized cell, according to Kohler and Milstein (1975) or to Buttin et al. (1978) for example.

According to another aspect of the invention, a monoclonal antibody as defined above is secreted by the hybridoma deposited under the Budapest Treaty at the CNCM (Collection Nationale de Culture de Microorganismes, Institut Pasteur, Paris, France) on Mar. 19, 2003, under accession number CNCM 1-2982. Such a monoclonal antibody binds to the natural HCV viral envelope and to the HCV E2 protein. Said monoclonal antibody is hereafter referred to as D4.12.9.

According to yet another aspect of the invention, a monoclonal antibody as defined above is secreted by the hybridoma deposited under the Budapest Treaty at the CNCM (Collection Nationale de Culture de Microorganismes, Institut Pasteur, Paris, France) on Mar. 19, 2003, under accession number CNCM 1-2983. Such a monoclonal antibody binds to the natural HCV viral envelope, to the natural HCV E2 protein, to the natural HCV E1 protein, is capable of precipitating the HCV E1E2 complex under its covalent form or its non covalent form, and is capable of binding to an epitope constituted of at least one or all of the above-defined sequences. Said monoclonal antibody is hereafter referred to as D32.10.

The heavy chain and the light chain of a D32.10 monoclonal antibody secreted by the hybridoma deposited under accession number CNCM 1-2983 were sequenced after purification of the antibody from a cell culture, as described in Example 5. The amino acid sequence of the full heavy chain of D32.10 is presented in SEQ ID NO: 34 and the corresponding DNA sequence is presented in SEQ ID NO: 54. The amino acid sequence of the full light chain of D32.10 is presented in SEQ ID NO: 46 and the corresponding DNA sequence is presented in SEQ ID NO: 55.

Consequently, the present invention provides an isolated antibody, or a functional fragment thereof, that comprises at least one heavy chain having an amino acid sequence selected from the group consisting of the amino acid sequence set forth in SEQ ID NO: 34, conservative variants thereof and antigen-binding regions thereof. In one particular embodiment, the isolated antibody or functional fragment thereof comprises at least one heavy chain comprising three CDRs having amino acid sequences set forth in SEQ ID NO: 43, SEQ ID NO: 44 and SEQ ID NO: 45 or conservative variants thereof. In another particular embodiment, the isolated antibody, or functional fragment thereof, comprises at least one heavy chain variable region having an amino acid sequence selected from the group consisting of the amino acid sequence set forth in SEQ ID NO: 35 and convervative variants thereof.

The present invention also provides an isolated antibody, or a functional fragment thereof, that comprises at least one light chain having an amino acid sequence selected from the group consisting of the amino acid sequence set forth in SEQ ID NO: 46, conservative variants thereof and antigen-binding regions thereof. In one particular embodiment, the isolated antibody or functional fragment thereof comprises at least one light chain comprising three CDRs having amino acid sequences set forth in SEQ ID NO: 51, SEQ ID NO: 52 and SEQ ID NO: 53 or conservative variants thereof. In another particular embodiment, the isolated antibody, or functional fragment thereof, comprises at least one light chain variable region having an amino acid sequence selected from the group consisting of the amino acid sequence set forth in SEQ ID NO: 55 and convervative variants thereof.

The invention further provides an isolated antibody, or a functional fragment thereof, that comprises at least one heavy chain having an amino acid sequence selected from the group consisting of the amino acid sequence set forth in SEQ ID NO: 34, conservative variants thereof and an antigen-binding regions thereof and at least one light chain having an amino acid sequence selected from the group consisting of the amino acid sequence set forth in SEQ ID NO: 46, conservative variants thereof and antigen-binding regions thereof. In one particular embodiment, the isolated antibody or functional fragment thereof comprising at least one heavy chain comprising comprising three CDRs having amino acid sequences set forth in SEQ ID NO: 43, SEQ ID NO: 44 and SEQ ID NO: 45 or conservative variants thereof, and at least one light chain comprising three CDRs having amino acid sequences set forth in SEQ ID NO: 51, SEQ ID NO: 52 and SEQ ID NO: 53 or conservative variants thereof. In another particular embodiment, the isolated antibody, or functional fragment thereof, comprises at least one heavy chain variable region having an amino acid sequence selected from the group consisting of the amino acid sequence set forth in SEQ ID NO: 35 and convervative variants thereof, and at least one light chain variable region having an amino acid sequence selected from the group consisting of the amino acid sequence set forth in SEQ ID NO: 47 and convervative variants thereof.

As used in the present context, the term “antibody” refers to an immunoglobulin molecular comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region. The heavy constant region is comprised of three domains. Each light chain is comprised of a light chain variable region (VL) and a light chain constant region. The light chain constant region is comprised of one domain. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementary determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

The terms “antigen-binding region”, “antigen-binding portion”, and “antigen-binding fragment” are used herein interchangeably. An antigen-binding portion of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen, in particular the ability to specifically bind to the natural HCV E1 protein and/or to the HCV E2 protein. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term “antigen-binding region” of an antibody include a Fab fragment, a F(ab′)2 fragment, a Fd fragment, a Fv fragment, a fragment which consists of a VH domain, a fragment which consists of a VL domain, and at least one isolated CDR. Furthermore, although the two domains of the Fv fragment, VL and VH are encoded by separate genes, they can be joined, using recombinant methods, by a synthetic linker resulting in a single polypeptide chain in which the VL and VH regions form a monovalent molecule (known as single chain Fv (ou scFv). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody. Other forms of single chain antibodies, such as diabodies, which are bivalent, bispecific antibodies, are also encompassed. These antibody fragments may be obtained using conventional techniques known to those of skilled in the art and the fragments may be screened for binding properties in the same manner as are intact antibodies.

In the present context, the terms “isolated” and “purified”, when used to characterize an antibody or antibody fragment, refers to an antibody or antibody fragment that is substantially free of other antibodies or antibody fragments having different antigenic specificities. Moreover, an isolated or purified antibody or antibody fragment is substantially free from other cellular material and/or chemicals and from components that normally accompany it in its native state or during its preparation.

As used herein, the term “conservative variant” of a polypeptide sequence refers to a variant of the polypeptide sequence that comprises at least one conservative sequence modification. “Conservative sequence modification” is intended to refer to an amino acid modification that does not significantly affect or alter the binding characteristics of the antibody (or antibody fragment) containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions or deletions. Modification can be introduced into an antibody of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.

“Conservative amino acid substitutions” of a residue in a reference sequence are substitutions that are physically or functionally similar to the corresponding reference residue, e.g., that have a similar size, shape, electric charge, chemical properties, including the ability to form covalent or hydrogen bonds, or the like. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues with the CDR regions of an antibody of the invention can be replaced with other amino acid residues from the same side chain family, and the altered antibody can be tested for retained binding ability using routine assays. Typically, no more than one, two, three, four or five residues within the CDR regions are conservally altered.

In certain embodiments, an antibody of the present invention is a polyclonal antibody. However, in certain preferred embodiments, an antibody of the present invention is a monoclonal antibody. As used herein, the term “monoclonal antibody” refers to a homogeneous antibody population involved in the highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies that typically include different antibodies directed to different antigenic determinants. Furthermore, the term “monoclonal antibody” refers to such monoclonal antibodies made in any number of manners including but not limited to by hybridoma, phage selection, recombinant expression, and transgenic animals.

In certain embodiments, an antibody of the present invention is a chimeric antibody. As used herein, the term “chimeric antibody” refers to an antibody wherein the amino acid sequence of the immunoglobulin molecule is derived from two or more species. Typically, the variable region of both light and heavy chains corresponds to the variable regions of antibodies derived from one species of mammals (e.g., mouse) with the desired specificity, affinity and capability while the constant regions are homologous to the sequences in antibodies derived from another (e.g., human) to avoid eliciting an immune response in that species.

In certain embodiments, an antibody of the present invention is a humanized antibody. As used herein, the term “humanized antibody” refers to forms of non-human (e.g., murine) antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof, that contain minimal non-human sequences. Typically, humanized antibodies are human immunoglobulins in which residues from the CDR are replaced by residues from the CDR of a non-human species (e.g., mouse) that have the desired specificity, affinity, and capability. In some instances, the Fv framework region (FR) residues of a human immunoglobulin are replaced with the corresponding residues in an antibody from a non-human species that has the desired specificity, affinity and capability. The humanized antibody can be further modified by the substitution of additional residues either in the Fv framework region and/or within the replaced non-human residues to refine and optimize antibody specificity, affinity and/or capability. In general, the humanized antibody will comprise substantially all of at least one, and typically two or three, variable domains containing all or substantially all of the CDR regions that correspond to the non-human immunoglobulin whereas all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humaniazed antibody can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Humanized antibodies may be produced by site-directed mutagenesis or chemical grafting or using recombinant methods.

In certain embodiments, an antibody of the present invention is a deimmunized antibody. Methods for the preparation of deimmunized antibodies are known in the art. Briefly, in such methods, the T-cell epitopes present in rodent antibodies can be modified by mutation (de-immunization) to generate non-immunogenic rodent antibodies that can be applied for therapeutic purposes in humans. Typically, deimmunized antibodies are created with human constant regions and by expression of genes encoding these antibodies in mammalian cells.

In yet another embodiment, an isolated antibody of the invention, or a functional fragment thereof, has heavy and light chain variable regions having amino acid sequences that are homologous to the amino acid sequences described herein, and wherein the antibody (or functional fragment thereof), retains the ability to specifically bind to the natural HCV E1 protein and/or to the natural HCV E2 protein.

For example, the invention provides an isolated antibody, or antigen-binding portion thereof, exhibiting the desired binding properties and comprising a heavy chain variable region and/or a light chain variable region, wherein the heavy chain variable region has an amino acid sequence that is at least 80% homologous to the amino acid sequence set forth in SEQ ID NO: 35 and wherein the light chain variable region has an amino acid sequence that is at least 80% homologous to the amino acid sequence set forth in SEQ ID NO: 47. For instance, the VH amino acid sequence may be 90%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO: 35, and the VL amino acid sequence may be 90%, 95%, 96%, 97%, 98% or 99% homologous to the sequence set forth in SEQ ID NO: 47. Such an antibody (or functional fragment thereof) may be obtained by mutagenesis (e.g., site-directed or PCR-mediated mutagenesis) of nucleic acid molecules encoding SEQ ID NO: 35 and/or SEQ ID NO: 47 by testing the encoded altered antibody (or fragment thereof) for retained binding properties.

As used herein, the term “homologous” is synonymous with the term “identity” and refers to the sequence similarity between two polypeptide molecules. When a position in both compared sequences is occupied by the same amino acid residue, then the respective molecules are homologous at that position. The percentage of homology between the two sequences corresponds to the number of matching or homologous positions shared by the two sequences divided by the number of positions compared and multiplied by 100. Generally, a comparison is made when two sequences are aligned to give maximum homology.

In another aspect, the present invention provides antigen-binding molecules that are capable of specifically binding to the natural HCV E1 protein and/or to the natural HCV E2 protein, but that are not antibodies.

In particular, the present invention provides an antigen-binding molecule comprising three complementary determining regions having amino acid sequences set forth in SEQ ID NO: 43, SEQ ID NO: 44 and SEQ ID NO: 45 or conservative variants thereof.

The present invention also provides an antigen-binding molecule comprising three complementary determining regions having amino acid sequences set forth in in SEQ ID NO: 51, SEQ ID NO: 52 and SEQ ID NO: 53 or conservative variants thereof.

The present invention further provides an antigen-binding molecule comprising three complementary determining regions having amino acid sequences set forth in SEQ ID NO: 43, SEQ ID NO: 44 and SEQ ID NO: 45 or conservative variants thereof, and three complementary determining regions having amino acid sequences set forth in in SEQ ID NO: 51, SEQ ID NO: 52 and SEQ ID NO: 53 or conservative variants thereof.

Such antigen-binding molecules may belong to any family of molecules except for the family of molecules defined herein as “antibodies”.

In another aspect, the present invention provides nucleic acid molecules that encode the antibodies of the invention. For example, the present invention provides a nucleic acid having a sequence selected from the group consisting of SEQ ID NO: 54, homologous variants thereof, and fragments thereof encoding antigen-binding portions of an antibody heavy chain. The invention also provides a nucleic acid having a sequence selected from the group consisting of SEQ ID NO: 55, homologous variants thereof, and fragments thereof encoding antigen-binding portions of an antibody light chain.

The nucleic acid may be present in whole cells, in a cell lysate, in a transgenic animal or may be nucleic acids in a partially purified or substantially pure form. A nucleic acid is “isolated” or “rendered substantially pure” when purified away from other cellular components or contaminants, e.g., other cellular nucleic acids or protein, for example using standard techniques such as alkaline/SDS treatment, CsCl banding, column chromatography, and agarose gel electrophoresis. A nucleic acid of the invention can be, for example, DNA or RNA and may or may not contain intronic sequences. In an embodiment, the nucleic acid is a cDNA molecule. The nucleic acid may be present in a vector such as a phage display vector, or in a recombinant plasmid vector.

Nucleic acids of the invention can be obtained using standard molecule biology techniques. Once DNA fragments encoding VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region genes to full-length antibody chain genes, to Fab fragment genes or to an scFv gene. In these manipulations, a VL- or VH-encoding DNA fragment may be operatively linked to another DNA molecule, or to a fragment encoding another protein, such as an antibody constant region or a flexible linker. The term “operatively linked”, as used in this context, is intended to mean that the two DNA fragments are joined in a functional manner, for example, such that the amino acid sequences encoded by the two DNA fragments remain in-frame, or such aht the protein is expressed under the control of a desired promoter.

The isolated DNA encoding the VH region may be converted to a full-length heavy chain gene by operatively linking the VH-encoding DNA to another DNA molecule encoding heavy chain constant regions. The sequences of human heavy chain constant region genes are known in the art and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be human IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region. For a Fab fragment heavy chain gene, the VH-encoding DNA can be operatively linked to another DNA molecule encoding only the heavy chain CH1 constant region.

The isolated DNA encoding the VL region can be converted to a full-length light chain gene (as well as to a Fab light chain gene) by operatively linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region. The sequences of human light chain constant region genes are known in the art and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or a lambda constant region.

To create a scFv gene, the VH- and VL-encoding DNA fragments are operatively linked to another fragment encoding a flexible linker such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VH and VL regions joined by the flexible linker.

Antibodies of the present invention can be produced by any of a wide variety of suitable methods known in the art. For example, antibodies may be produced in a host cell transfectoma using, for example, a combination of recombinant DNA techniques and gene transfection methods, as is well known in the art. For example, to express antibodies, or fragments thereof, DNAs enconding partial or full-length light and heavy chains, can be obtained by standard molecular biology techniques (e.g., PCR amplification, or cDNA cloning using a hybridoma that expresses the antibody of interest) and the DNAs can be inserted into expression vectors such that the genes are operatively linked to transcriptional and translational control sequences. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors or, more typically, both genes may be inserted into the same expression vector. The antibody genes may be inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present). The light and heavy chain variable regions described herein can be used to create full-length antibody genes of any antibody isotype by inserting them into expression vectors already encoding heavy chain constant and light chain constant regions of the desired isotype such that the VH segment is operatively linked to the heavy chain constant region and the VL segment is operatively linked to the light chain constant region within the vector. Alternatively or additionally, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (e.g., a signal peptide from a non-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expression vectors of the invention carry regulatory sequences that control the expression of the antibody chain genes in a host cell. The term “regulatory sequence” is intended to include promoters, enhancers and other suitable expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. It will be appreciated by one skilled in the art tha the design of an expression vector, including the selection of regulatory sequences, may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. Regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus (e.g., the adenovirus major late promoter), and polyoma. Alternatively, non viral regulatory sequences may be used, such as the ubiquitin promoter or P-globin promoter.

In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced. For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Selectable marker genes include, but are not limited to, the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cell with methotrexate selection/amplification) and the neo gene (for G418 selection).

For expression of the light and heavy chains, the expression vector(s) encoding the heavy and light chains is introduced by transfection into a host cell by standard techniques. As used herein, the term “transfection” is intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell. Such techniques include, but are not limited to, electroporation, calcium-phosphate precipitation, DEAE-dextran transfection, and the like. In certain preferred embodiments, expression of antibodies of the invention are carried out in eukaryotic cells, in particular mammalian host cells, which are likely than prokaryotic cells to assemble and secrete a properly folded and immunologically active antibody.

Mammalian host cells suitable for expressing the recombinant antibodies of the invention include, but are not limited to, Chinese Hamster Ovary (CHO cells), NSO myeloma cells, COS cells and SP2 cells. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can then be recovered from the culture medium using standard protein purification methods.

In another aspect, the present invention provides bispecific molecules comprising an antibody, or fragment thereof, of the invention. An antibody of the invention, or antigen-binding portions thereof, can be derivatized or linked to another functional molecule, e.g., another peptide or protein such as another antibody or ligand for a receptor) to generate a bispecific molecule that binds to at least two different sites or target molecules (e.g., two different sites on HCV). The antibody of the invention may in fact be derivatized or linked to more than one other functional molecule to generate multi-specific molecules that bind to more than two different binding sites, and/or target molecules. Such multi-specific molecules are also intended to be encompassed by the term “bispecific molecule”, as used herein. To create a bispecific molecule according to the present invention, an antibody of the invention can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic.

Accordingly, the present invention includes bispecific molecules comprising at least one first binding specificity for the natural HCV E1 and E2 proteins and a second binding specificity for a second target epitope.

In one embodiment, a bispecific molecule of the present invention comprises as a binding specificity at least one antibody, or an antibody fragment thereof, including, e.g., a Fab, Fab′, F(ab′)2, Fv, or a single chain Fv. The antibody may also be a light chain or a heavy chain dimer, or any minimal fragment thereof, such as a Fv or a single chain construct. Antibodies that can be employed in the bispecific molecules may be murine, chimeric, or humanized monoclonal antibodies.

A bispecific molecule of the present invention can be prepared by conjugating the constituent binding specificities using methods known in the art. For example, each binding specificity of the bispecific molecule can be generated separately and then conjugated to one another. When the binding specificities are proteins or peptides, a variety of coupling or cross-linking agents can be used for covalent conjugation. Conjugating agents are well known in the art, and are for example commercially available from Pierce Chemical Co. When the binding specificies are antibodies, they can be conjugated by sufhydryl bonding of the C-terminus hinge regions of the two heavy chains. Alternatively, both binding specificities can be encoded in the same vector and expressed and assembled in the same host cell.

Binding of a bispecific molecule to its specific targets can be confirmed by, for example, ELISA, radioimmunoassay, FACS analysis, or Western Blot assay.

II—Hybridomas

According to another aspect, the invention relates to a hybridoma deposited under the Budapest Treaty at the CNCM (Collection Nationale de Culture de Microorganismes, Institut Pasteur, Paris, France) on Mar. 19, 2003, under accession number CNCMI-2983.

According to yet another aspect, the invention relates to a hybridoma deposited under the Budapest Treaty at the CNCM (Collection Nationale de Culture de Microorganismes, Institut Pasteur, Paris, France) on Mar. 19, 2003, under accession number CNCMI-2982.

III—Compositions and Methods of Use

The present invention also provides pharmaceutical compositions comprising as active substance at least one of the antibodies, or at least one fragment thereof, as defined above and at least one pharmaceutically acceptable excipient or vehicle. Pharmaceutically acceptable excipients or vehicles suitable for use in the present invention are for example described in Remington's Pharmaceutical Sciences 16th ed./Mack Publishing Co. Other compounds, in particular antiviral compounds can be included in a pharmaceutical composition according to the invention, such as, for example, other anti-HCV antibodies and fragments or derivatives thereof, interferons, RNA polymerase inhibitors, protease inhibitors and/or helicase inhibitors.

A pharmaceutical composition according to the present invention may be administered using any suitable dosage and/or mode of administration. For example, a pharmaceutical composition may be administered in a single dose or multiple doses. In case of a single dose, the composition may comprise from about 0.1 mg of antibody per kg of body weight, to about 1 g of antibody per kg of body weight, in particular from about 1 mg/kg to about 100 mg/kg. In case of multiple doses, the composition can be administered in a dosage of about 0.1 mg of antibody per kg of body weight per day, to about 1 g of antibody per kg of body weight per day, in particular from about 1 mg/kg/day to about 100 mg/kg/day.

The present invention also provides for the use of at least one of the antibodies, or fragments thereof, as defined above, for the preparation of a medicament for the diagnostic, the prevention and/or the treatment of HCV infections.

The antibodies according to the invention, or fragments thereof, can be used for the detection of HCV viral particles in biological samples suspected to contain HCV. Such samples may be obtained from patients infected by HCV or at risk of being infected by HCV and may be, for example, samples of blood, plasma or serum. Any of the methods well known in the art for the detection of viruses using antibodies, such as ELISA or EIA, can be applied with the antibodies of the invention.

The antibodies according to the invention, or fragments thereof or pharmaceutical compositions thereof, may be administered to patients infected or not with HCV, with the goal of to neutralizing HCV infection. Without wishing to be bound by theory, it may be thought that HCV infection neutralization can proceed by preventing HCV from contacting target cells, for instance through binding of the antibody, or fragment thereof, to envelope molecules, such as E1 and/or E2, known to bind to target cells membrane receptors, thereby preventing HCV-target cells binding.

The antibodies according to the invention, or fragments thereof or pharmaceutical compositions thereof, may be administered to patients infected by HCV to prevent HCV viral particles from infecting cells, in particular hepatocytes, or to promote capture of HCV coated with said antibodies or fragments by cells of the immune system. In particular, the antibodies, or fragments or pharmaceutical compositions thereof, may be administered to patients which are to receive a transplanted organ, for example a liver, prior, during or after the transplantation surgery, to neutralize HCV viral particles which may be contained in the transplanted organ.

IV—Enveloped Viral Particles and Compositions Thereof

The invention also relates to enveloped viral particles that are capable of binding to at least one of the antibodies, or fragments thereof, defined above. As used herein, the expression “enveloped viral particle” refers in particular to a HCV virion, or part of a HCV virion, which contains an envelope, the envelope comprising lipids and/or proteins, in particular glycoproteins, associated in leaflets. The invention also relates to an antibody which binds to an enveloped viral particle capable of binding to at least one of the antibodies as defined above.

According to another aspect, the invention relates to a composition of HCV viral particles derived from samples of human blood, plasma or sera, wherein the concentration of HCV RNA copies is about 100 to 1000 fold higher than the concentration of HCV RNA copies in the initial samples of human, blood, plasma or sera from which it is derived, and is in particular higher than about 107 copies/ml. The high concentration of the compositions according to the invention allows efficient immunization of animals using these compositions. As used herein, the term “HCV RNA” refers more particularly to HCV genomic RNA. The HCV RNA contents of a sample can be measured by RT-PCR, in particular quantitative RT-PCR, using the Amplico™ HCV Monitor™ Roche Diagnostics (Young et al., 1993) for example or any other suitable similar assay. Alternatively, the HCV RNA contents of a sample can be measured using the NASBA (Nucleic Acid Sequence Based Amplification) technology (Damen et al., 1999). The HCV RNA contents of a sample can be expressed in terms of number of copies of HCV RNA molecules in said sample, one copy being equivalent to one International Unit (UI). The HCV RNA content of a sample is indicative of the quantity HCV virions contained in said sample.

As mentioned above, a composition containing more than about 107 copies/ml of HCV RNA allows for an efficient immunization of animals using said composition. Accordingly, in certain preferred embodiments, the invention more specifically relates to a composition as defined above, wherein the number of HCV RNA copies is from about 108 to about 109 UI per mg of protein. The protein content of a composition may be assessed by methods well known to those skilled in the art, for example using the Lowry assay (Lowry et al., 1951) such as the Biorad protein assay (Biorad Laboratories). This measure represents the specific activity of the composition, it is indicative of the purity of the composition; the higher the number of HCV RNA copies per mg of protein the higher the purity of the HCV containing composition.

The invention further relates to a composition as defined above, wherein the volume of said composition is from about 0.1 ml to about 10 ml. A composition volume of about 0.1 ml to about 10 ml corresponds to a composition as defined above containing at least 106 HCV RNA copies. Such a composition is useful for the immunization of animals. For example, the efficient immunization of a mouse requires the administration of a HCV viral particles composition containing more than about 106 HCV RNA copies, as described in the Examples below.

The invention also relates to isolated HCV enveloped subviral particles substantially devoid of HCV RNA and of HCV core protein. As used in this particular context, the term “isolated” means that the particles have been extracted from their natural environment, in particular separated from other HCV viral particles or parts thereof which contain HCV RNA and/or HCV core protein. The expression “substantially devoid of HCV RNA and of HCV core protein” means that the solution containing the above defined subviral particles contains less than 104 UI/ml of HCV RNA as measured the Amplicor™ HCV Monitor™ Roche Diagnostics assay (Young et al., 1993), and less than 1 pg/ml of core protein as measured with the Ortho-Clinical Diagnostics test (Aoyagi et al., 1999). The presence of the envelope can be assessed for instance by an EIA or ELISA test with the antibody secreted by the hybridoma deposited at the CNCM under accession number 1-2983 (i.e., a D32.10 monoclonal antibody), or the antibody secreted by the hybridoma deposited at the CNCM under accession number 1-2982 (i.e., a D4.12.9 monoclonal antibody).

As used herein, the expression “HCV enveloped subviral particle” means that the particle contains substantially only the envelope part of the HCV virion, i.e., the lipids and proteins, in particular glycoproteins, associated in leaflets. HCV viral particles isolated thus far either contained HCV RNA and/or HCV core protein.

The invention more specifically relates to isolated HCV enveloped subviral particles as defined above, which can bind to any of the antibodies described herein. Binding of a subviral particle to an antibody of the invention may be assessed using any of several methods well known to those skilled in the art, such as immunoprecipitation, ELISA, EIA or Western blotting for example.

According to another embodiment, the invention relates to a composition comprising purified HCV enveloped complete viral particles, wherein said purified HCV enveloped complete viral particles contain HCV RNA, HCV core protein and HCV envelope, and can bind to any of the antibodies described herein. The invention more particularly relates to a composition comprising purified natural HCV enveloped complete viral particles. As used herein, the expression “HCV enveloped complete viral particles” means that HCV virions contain HCV genomic RNA, HCV core protein and HCV envelope. There has been no report in the prior art of a HCV viral particle containing these three components. The presence of HCV RNA, HCV core protein and HCV envelope can be assessed using the methods mentioned above. As used in this context, the term “purified” means a HCV enveloped complete viral particle of the invention has been separated from other compounds, such as HCV enveloped subviral particles. In particular, it can be assessed, for example by electron microscopy, that the composition contains 90% less HCV enveloped subviral particles than HCV enveloped complete viral particles. Binding of a HCV enveloped complete viral particle to the antibodies an antibody of the invention can be assessed using any of the several methods well known to those skilled in the art, such as immunoprecipitation, ELISA, EIA or Western blotting for example.

According to another embodiment, the invention relates to a process for preparing a composition of HCV viral particles comprising the following steps: at least two ultracentrifugations of a sample resulting from a clarified plasmapheresis of a HCV infected patient to obtain a HCV enriched pellet; resuspension of the HCV enriched pellet in an aqueous solution to obtain a composition of HCV viral particles. The ultracentrifugations may be carried out for instance at a speed of about 190,000 g to about 220,000 g, preferably 210,000 g, during about 3 hours to about 5 hours, preferably 4 hours. The preferred centrifugation conditions lead to precipitation of viral particles, such as HCV viral particles, while other compounds contained in the clarified plasma are not precipitated. The term “plasmapheresis” means that the patient's blood has been filtered to obtain plasma while the remainder of the blood has been re-injected to the patient. The term “clarified”, as used herein to characterize a plasmapheresis, means that the plasma obtained from the plasmapheresis has been centrifuged at low speed, in particular at 3000 g during 30 minutes. The initial volume of plasmapheresis before ultracentrifugation is advantageously of about 1 litre.

According to another embodiment, the invention relates to a composition of HCV viral particles such as obtained according to the above-mentioned process for preparing a composition of HCV viral particles.

According to yet another embodiment, the invention relates to a process for preparing a composition of HCV enveloped subviral particles comprising the following steps: at least two ultracentrifugations of a sample resulting from a clarified plasmapheresis of a HCV infected patient to obtain a HCV enriched pellet; resuspension of the HCV enriched pellet in an aqueous solution; ultracentrifugation of the resuspended HCV enriched pellet in a sucrose density gradient to separate the elements of the resuspended HCV enriched pellet into fractions according to their density; recovery of the fractions containing substantially no HCV RNA, substantially no HCV core protein and containing particles capable of binding to a monoclonal antibody described herein such as D32.10 and/or D4.12.9, to obtain a composition of HCV enveloped subviral particles. The ultracentrifugation of the resuspended HCV enriched pellet in a sucrose density gradient maybe carried out from about 190,000 g to about 220,000 g, preferably 210,000 g, during about 40 hours to about 50 hours, preferably 48 hours; the sucrose gradient can be advantageously from about 10% to about 60% w/w, from about 20 to about 50% w/w, from about 25% to about 45% w/w, or from about 30% to about 45% w/w.

All the fractions obtained are tested for the presence of HCV RNA, HCV core protein and/or particles capable of binding to D32.10 or D4.12.9. The fractions are said to contain substantially no HCV RNA when the content of HCV RNA as measured by Amplicor™ HCV Monitor™, Roche Diagnostics assay (Young et al., 1993), is less than about 104 UI/ml. The fractions are said to contain substantially no HCV core protein when the content in HCV core protein as measured by the Ortho-Clinical Diagnostics test (Aoyagi et al., 1999) is less than about 1 pg/ml. In certain embodiments, the monoclonal antibody used is the monoclonal antibody D32.10 which is secreted by the hybridoma deposited at the CNCM under accession number 1-2983. The particles are said to be capable of binding to the above mentioned monoclonal antibody if they can be tested positive in the EIA test described below.

In this EIA test, polystyrene plates of 96-wells (Falcon; Becton Dickinson France S. A, Le Pont de Claix) are coated with the different fractions diluted from 10−1 to 10−4. The plates are incubated overnight at 4° C. and are then saturated with Tris-NaCl (TN) buffer (20 mM Tris-HCl, pH 7.5 and 100 mM NaCl) containing 5% (w/v) bovine serum albumin (BSA). The D32.10 mAb diluted in a mixture of TN/BSA buffer and 50% normal human serum (NHS) at a concentration of 5 μg/ml is added to each well and incubated for 2 hours at 37° C. The bound antibody is detected with a horseradish peroxidase (HRPO)-conjugated F(ab′)2 fragment of anti-mouse immunoglobulins (diluted 1/5,000; Immunotech) and with orthophenylenediamine (OPD) and hydrogen peroxide (H2O2) as substrates. Optical density (OD) is determined at 450 nm or at 490 nm with an ELISA plate reader (MRX, Dynex). The results are considered as positive when higher than a cut-off value corresponding to the mean of negative controls multiplied by 2.1. The recovered fractions have in particular a sucrose density of approximately 1.13 to 1.15 g/ml. Alternatively, the fractions may be first tested for the presence of particles capable of binding to D32.10 and/or D4.12.9: if substantially no such particles are present, then no other test is performed; if such particles are present, then the HCV RNA test and/or the HCV core protein test are performed.

According to another embodiment, the invention relates to a composition of HCV enveloped subviral particles such as obtained according to the above process for preparing a composition of HCV enveloped subviral particles.

According to yet another embodiment, the invention relates to a process for preparing a composition of purified HCV enveloped complete viral particles comprising the following steps: at least two ultracentrifugations of a sample resulting from a clarified plasmapheresis of a HCV infected patient to obtain a HCV enriched pellet; resuspension of the HCV enriched pellet in an aqueous solution; ultracentrifugation of the resuspended HCV enriched pellet in a sucrose density gradient to separate the elements of the resuspended HCV enriched pellet into fractions according to their density; recovery of the fractions containing from about 5.105 to about 106 UI of HCV RNA per ml, from about 50 to about 100 pg of HCV core protein per ml, and containing particles capable of binding to a monoclonal antibody described herein such as D32.10 and/or D4.12.9, to obtain a composition of purified HCV enveloped complete viral particles. The ultracentrifugation of the resuspended HCV enriched pellet in a sucrose density gradient may be carried out from about 190,000 g to about 220,000 g, preferably 210,000 g, during about 40 hours to about 50 hours, preferably 48 hours; the sucrose gradient can be advantageously from about 10% to about 60% w/w, from about 20 to about 50% w/w, from about 25% to about 45% w/w, or from about 30% to about 45% w/w. The HCV RNA content, HCV core protein content and the binding capability of the particles may be measured as indicated above. In certain embodiments, the monoclonal antibody used is the D32.10 monoclonal antibody which is secreted by the hybridoma deposited at the CNCM under accession number 1-2983.

The composition obtained using this process contains purified HCV enveloped complete viral particles and is substantially devoid of HCV enveloped subviral particles. In particular, it can be assessed, for example by electron microscopy, that the composition contains 90% less HCV enveloped subviral particles than HCV enveloped complete viral particles. The recovered fractions have a sucrose density of approximately 1.17 to 1.21 g/ml, more particularly of approximately 1.17 to 1.20 g/ml, of approximately 1.19 to 1.21 g/ml, of approximately 1.17 to 1.19 g/ml, or of approximately 1.19 to 1.20 g/ml. Alternatively, the fractions may first be tested for the presence of HCV RNA: if more than 105 copies/m1 of HCV RNA are present, then the HCV core protein test is performed; if more than 50 pg/ml of core protein are present, then the presence of particles capable of binding to D32.10 and/or to D4.12.9 is tested; if less than 105 copies/ml of HCV RNA are present then no other test is performed.

According to another embodiment, the invention relates to a composition of purified HCV enveloped complete viral particles obtained according to the process described above.

The invention also relates to a process for preparing a monoclonal antibody secreted by the hybridoma deposited at the CNCM under accession number I-2983, comprising the following steps: immunizing an animal, in particular a mammal, with a composition of HCV viral particles of the invention, or such as prepared according to the invention, and recovering the generated antibodies; selecting, among the generated antibodies, monoclonal antibodies on their ability to bind to the HCV viral particles contained in the composition of HCV viral particles. The composition of HCV viral particles used in this process can be obtained as follows: at least two ultracentrifugations of a sample resulting from a clarified plasmapheresis of a HCV infected patient to obtain a HCV enriched pellet; resuspension of the HCV enriched pellet in an aqueous solution to obtain a composition of HCV viral particles. The selection of the generated antibodies can be carried out with an indirect EIA test as defined above, the plates being coated with the composition of HCV viral particles of the invention.

According to yet another embodiment, the invention relates to a process for preparing a monoclonal antibody secreted by the hybridoma deposited at the CNCM under accession number I-2982, comprising the following steps: immunizing an animal, in particular a mammal, with a composition of purified HCV enveloped complete viral particles of the invention, or such as prepared according to the invention, and recovering the generated antibodies; selecting, among the generated antibodies, monoclonal antibodies on their ability of binding to the purified HCV enveloped complete viral particles contained in the above mentioned composition of purified HCV enveloped complete viral particles. The composition of purified HCV enveloped complete viral particles used in this process can be obtained as follows: at least two ultracentrifugations of a sample resulting from a clarified plasmapheresis of a HCV infected patient to obtain a HCV enriched pellet; resuspension of the HCV enriched pellet in an aqueous solution; ultracentrifugation of the resuspended HCV enriched pellet in a sucrose density gradient to separate the elements of the resuspended HCV enriched pellet into fractions according to their density; recovery of the fractions containing from about 5.105 to about 106 UI of HCV RNA per ml, from about 50 to about 100 pg of HCV core protein per ml, and containing particles capable of binding to a monoclonal antibody described herein such as D32.10 or D4.12.9, to obtain a composition of purified HCV enveloped complete viral particles. The selection step can proceed as described above.

According to another embodiment, the invention relates to a process for preparing a monoclonal antibody directed against HCV, in particular HCV enveloped subviral particles, comprising the following steps: immunizing an animal, in particular a mammal, with a composition of HCV enveloped subviral particles of the invention, or prepared according to the invention, and recovering the generated antibodies; selecting, among the generated antibodies, monoclonal antibodies on their ability of binding to the HCV enveloped subviral particles contained in the above mentioned composition of HCV enveloped subviral particles. The composition of HCV enveloped subviral particles used in this process can be obtained as follows: at least two ultracentrifugations of a sample resulting from a clarified plasmapheresis of a HCV infected patient to obtain a HCV enriched pellet; resuspension of the HCV enriched pellet in an aqueous solution; ultracentrifugation of the resuspended HCV enriched pellet in a sucrose density gradient to separate the elements of the resuspended HCV enriched pellet into fractions according to their density; recovery of the fractions containing substantially no HCV RNA, substantially no HCV core protein and containing particles capable of binding to a monoclonal antibody described herein such as D32.10 or D4.12.9, to obtain a composition of HCV enveloped subviral particles. The selection step can proceed as described above.

The present invention also relates to antibodies directed against the HCV enveloped subviral particles of the invention.

The invention further relates to a pharmaceutical composition comprising as active substance at least one antibody directed against HCV enveloped subviral particles and a pharmaceutically acceptable excipient or vehicle.

According to another embodiment, the invention relates to a pharmaceutical composition comprising as active substance the HCV enveloped subviral particles described herein, or a composition comprising the HCV enveloped subviral particles described herein, and a pharmaceutically acceptable excipient or vehicle vehicle.

Additionally adjuvants, such as defined for instance in Remington's Pharmaceutical Sciences 16th ed./Mack Publishing Co. can be added to the pharmaceutical composition, such as for instance incomplete Freund's adjuvant, aluminum salts or aluminum hydroxide.

Such compositions can be administrated, for instance, in a single dose comprising from 1 mg to 1 g of HCV enveloped subviral particles.

According to another embodiment, the invention relates to the use of the HCV enveloped subviral particles described herein, or of a composition thereof, to induce an immune reaction against the HCV enveloped subviral particles or against HCV the enveloped complete viral particles. As used herein, the expression “induce an immune reaction” means that B cells secreting antibodies directed against HCV viral particles can be activated or that T cells destroying cells infected by HCV can be activated.

According to another embodiment, the invention relates to the use of the HCV enveloped subviral particles described herein, or of a composition thereof, for the preparation of a medicament for the diagnostic, the prevention and/or the treatment of HCV infections. Thus, for example, the HCV enveloped subviral particles according to the invention can be used to assess the presence of antibodies directed against HCV in immunoassays according to methods well known those skilled in the art, such as EIA, ELISA. Alternatively or additionally, the HCV enveloped subviral particles according to the invention can be used for the preparation of a vaccine against hepatitis C, in particular a therapeutic vaccine. As used herein, the expression “therapeutic vaccine” means that the vaccine is capable of improving the condition of patient infected by HCV, for example by inducing the production of antibodies directed against HCV.

EXAMPLES

Example 1

Monoclonal Antibody Directed Against the Natural HCV Viral Envelope

HCV Viral Particles Preparation. To obtain virus materials in good supply, the purification of HCV viral particles was performed from plasmaphereses. The selected patient developed chronic active hepatitis C after partial liver transplantation for C viral cirrhosis, and 12 months later multiple myeloma associated with hypergammaglobulinemia which was the cause of plasma exchange. The patient showed abnormal elevated serum aminotransferase (ALT/AST) levels and was positive for anti-HCV antibodies. The patient was negative for all HBV and HIV markers. The HCV RNA content of initial material was 6×105 copies/ml (Amplicor™ HCV Monitor™ Roche Diagnostics, Meylan, France), and the genotype 1b (INNO-LIPA assay, Innogenetics, Gent, Belgium).

Clarified plasmapheresis was used to prepare a HCV-enriched pellet by two successive ultracentrifugations at 210,000 g for 4 h at 4° C. The final pellet was resuspended in 1 ml of Tris-NaCl-EDTA (TNE) buffer (20 mmol/L Tris-HCl, pH 7.5, 100 mmol/L NaCl, mmol/L EDTA) (concentration 240 fold), and stored at −80° C. The HCV RNA content of this material was 3×107 copies/ml (Monitor, Roche) and the protein concentration 5 mg/ml (i.e. about 107 copies of HCV RNA per mg of protein).

Hybridoma Preparation. In order to generate monoclonal antibodies (mAbs), BALB/c mice were inoculated with 100 μg (106 copies of HCV RNA) of HCV-pelleted material in complete Freund adjuvant, followed 1 week later with 100 μg of virus in incomplete Freund adjuvant. The three immunized mice developed high serum antibody titers against HCV, detected by indirect enzyme immunoassay (EIA). Three weeks later, the mice were boosted with 50 μg of virus in phosphate buffered saline (PBS). After 5 days the injection was repeated and 3 days later the spleen was removed for fusion with x63 myeloma cells using the procedure described by Buttin et al. (1978). Hybridoma culture supernatants were screened for the presence of HCV-specific antibodies by indirect EIA using the immunogen as the solid phase (5 μg/ml) and peroxidase-conjugated F(ab′)2 fragment of anti-mouse immunoglobulins (Amersham, France) as a revelation secondary antibody (Petit et al., 1987). The diluent contained 50% normal human serum (NHS) to eliminate non-specific reactivity directed against NHS proteins possibly associated with HCV viral particles. The hybrids giving the strongest signal (P/N>10) to HCV were then recloned by limiting dilution and their specificity further determined. Seven clones (C9.19.16, C2.22.1, D32.10, D3.20.12, C7.24.19, C7.14.41 and C1.9.3) were selected and four (D32.10, C2.22.1, C9.19.16 and D3.20.12) were propagated by injection into pristane-primed BALB/c mice for ascitic fluid production, and then purified by precipitation with 50% saturated (NH4)2SO4 followed by affinity chromatography in Sepharose-Protein G (Pharmacia, France). Isotype was determined by ELISA with methods well known to the man skilled in the art. The seven clones analyzed gave antibodies of the IgG1 isotype.

Monoclonal Antibody Characterization. Indirect EIA was used to evaluate the interaction of the above described monoclonal antibodies with the viral particles. Polystyrene plates of 96-wells (Falcon; Becton Dickinson France S. A, Le Pont de Claix) were coated with the HCV preparation (1 mg of protein per ml) diluted from 10−1 to 10−6 (corresponding to 100 μg/ml to 1 ng/ml). The plates were incubated overnight at 4° C. and were then saturated with Tris-NaCl (TN) buffer (20 mM Tris-HCl, pH 7.5 and 100 mM NaCl) containing 5% (w/v) bovine serum albumin (BSA). The D32.10 mAb diluted in a mixture of TN/BSA buffer and 50% normal human serum (NHS) at a concentration of 5 μg/ml was added to each well and incubated for 2 h at 37° C. The bound antibody was detected with the horseradish peroxidase (HRPO)-conjugated F(ab′)2 fragment of anti-mouse immunoglobulins (diluted 1/5,000; Immunotech) and with orthophenylenediamine (OPD) and hydrogen peroxide (H2O2) as substrates. Optical density (OD) was determined at 450 nm with an ELISA plate reader (MRX, Dynex). The results were considered as positive when higher than a cut-off value corresponding to the mean of negative controls multiplied by 2.1. The seven antibodies obtained did recognize the viral particles. To establish the native polypeptide specificity of the mAbs, immunoblotting using the immunogen as antigenic probes (Petit et al., 1987) was carried out. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) on 12.5% gels was performed under reducing conditions (2% SDS+5% 2-ME). After protein transfer onto PVDF membranes, mAbs (2 to 5 μg/ml) were tested as primary antibodies, diluted in 50% NHS, and IgGs bound were detected by incubation with peroxydase-conjugated (Fab′)2 fragment of anti-mouse immunoglobulins (diluted 1/10,000: IMMUNOTECH), as secondary antibody. The bands were visualized by enhanced chemoluminescence (ECL+) system from Amersham.

All mAbs tested, except for D32.10, gave negative reactions with HCV polypeptides under reducing conditions. It is worth noting that the mAbs selected were not reactive with either human serum albumin or with γ- or μ-chains of immunoglobulins (Ig), except for C1.9.3 and C7.24.19 which slightly reacted with human IgG in EIA (approximately 5-fold the negative value).

The polypeptide specificity of the seven antibodies was also examined by immunoprecipitation. HepG2 cells infected by vaccinia virus recombinants expressing HCV proteins (E1, E2 or E1E2) were metabolically labeled with 35S-Translabel (ICN) as previously described (Dubuisson et al., 1996). Cells were lysed with 0.5% NP-40 in 10 mM Tris-HCl (pH7.5), 150 mM NaCl, and 2 mM EDTA. The precipitates were treated with Laemmli sample buffer and analyzed by SDS-PAGE under reducing or non-reducing conditions (FIG. 1). The monoclonal antibodies tested did not show non specific reactivity directed against cellular components, except for C9.19.16 (>200 kDa), D3.20.12 (three intense bands at 70, 50 and 46 kDa) and more faintly, C2.22.1 (70, 50, 46 kDa) and D32.10 (multiple very faint bands) (FIG. 1(A), control vector). All antibodies specifically immunoprecipitated E1 (FIG. 1(B) and E2 (FIG. 1(C)), when the two HCV glycoproteins were expressed separately, as well as E1E2 heterodimers (FIG. 1(D)), when these proteins were co-expressed. Interestingly, the pattern was different after SDS-PAGE under non-reducing and reducing conditions (FIGS. 1(D) and (E)). All recognized disulfide-linked E1E2 aggregates, which were detected in the upper part of the gels under non-reducing conditions (FIG. 1(E)). One mAb, D32.10, was found to react mostly with aggregates and also with E1E2 non covalently-linked mature complex (FIG. 1(E), non-reducing conditions). All mAbs did not recognize denatured recombinant E1 or E2 proteins expressed in heterologous system by Western blot analysis, suggesting they recognized conformational epitopes on HCV viral particles. Collectively, these results indicate that these mAbs specifically recognize disulfide-linked E1E2 complexes expressed at the surface of natural serum-derived HCV viral particles. It is highly probable that they react with a shared conformational epitope between E1 and E2 proteins. Monoclonal antibody D32.10, which interacts with the E1E2 complex under its covalent form or non-covalent form was chosen for further studies.

Monoclonal Antibody D32.10 Antigen Mapping. To test the HCV type specificity of D32.10, an indirect EIA was carried out, according to the procedure already described, with four different HCV preparations (1 mg of protein per ml) diluted from 10−1 to 10−6 (corresponding to 100 μg/ml to 1 ng/ml). In addition to the immunogen HCV preparation, a preparation obtained from the same patient was used, as well as two other preparation obtained from two different patients with chronic hepatitis C, one of the patient having been found to carry to distinct genotypes in serum, namely HCV1 a and HCV2a. D32.10 was found able to recognize all four of the HCV preparations, thereby indicating that it is able of recognizing determinants not restricted to the 1b genotype of the immunogen.

To assess the native polypeptide specificity of D32.10 a Western blot analysis was carried out. Untreated HCV-enriched pellet was used as the antigenic probe, at different concentrations, varying from 0.1 to 1 mg/ml. The antigen was subjected to SDS-PAGE on 12.5% gels under reducing or non-reducing conditions (2% SDS±5% 2-mercaptoethanol (2-ME)). After protein transfer onto PVDF membranes, immunoblotting was performed using mAb D32.10 (2 to 5 μg/ml) as primary antibody, diluted in 50% NHS. Mouse IgGs bound were then detected by incubation with peroxidase-conjugated (Fab′)2 fragment of anti-mouse immunoglobulins (diluted 1/10,000; DAKO), as secondary antibody. Protein bands were visualized by enhanced chemiluminescence (ECL+) system from Amersham. Glycosidase digestion was performed as previously described by Sato et al. (1993) on circulating HCV virions. The HCV-enriched pellet (HCV-L, 4 μg) was treated with 5, 10 or 20 mU/ml of glycosidase A (peptide-N-glycosidase A or PNGase A; ROCHE) in 100 mM citrate/phosphate buffer (pH 6.0) for 18 h at 37° C. Deglycosylation of purified HCV viral particles was also performed by incubation overnight at 37° C. in 50 mM sodium acetate buffer (pH 5.5) containing endoglycosidase H (endo H, 5 mU/μl from Roche), 20 mM dithiothreitol (DTT) and 0.1% Triton X100. The control digestion was performed using the same conditions as for the PGNase A digestion or the endo H digestion, except that the enzyme was omitted. Samples were then treated with electrophoresis sample buffer containing reducing agent and analyzed by SDS-PAGE.

The results of the Western blot experiment are presented in FIG. 2. When two concentrations of the same sample (2.5 and 5 μg, lanes 2 and 3, respectively) were analyzed under reducing conditions (2% SDS/5% 2-ME), mAb D32.10 recognized a major band of 68 kDa and another band of 31 kDa, corresponding to E2 and E1 respectively. However, under non-reducing conditions, mAb 32.10 recognized disulfide linked complexes recovered in the upper part of the gel (>200 kDa). These high molecular weight bands (FIG. 2, lane 1) very probably correspond to heterodimeric E1E2 complexes.

Asparagine-linked complex type sugar chains have been shown to be present on the surface of native virions of HCV (Sato et al., 1993), thus the ability of D32.10 mAb to recognize HCV-specific proteins after treatment of the HCV preparation with glycosidase (PNGase A) at different concentrations (20, 10 and 5 mU/ml) was examined. As shown in FIG. 3A (lanes 1, 2 and 3, respectively), D32.10 reacted with deglycosylated forms of El, especially the 25 kDa species, which accumulated at the highest concentration of the enzyme. While E1-related products could be clearly detected by D32.10 after deglycosylation, the E2-related products were not clearly identified after the treatment. Endoglycosidase H (endo H) digestion allowed the investigation of the sensitivity of E1E2 complexes expressed on natural HCV viral particles to endo H cleavage. As shown in FIG. 3(B), a shift in molecular weight was observed for both E2 (from 68 to 42 kDa) and E1 (from 34 to 24 kDa) proteins suggesting that E1 and E2 possess a complex glycosylation on serum-derived native HCV viral particles, accounting for partial endo H resistance, such as a mixture of endo H resistant complex glycans and sensitive forms.

D32.10 Epitope Mapping

a. Screening of a Peptide Library. To further characterize the epitope recognized by mAb D32.10, the antibody was used to screen a dodecapeptide phage display library.

A Ph.D.12™ Phage Display Peptide Library Kit was obtained from New England BioLabs Inc. This is a combinatorial peptide 12-mers fused to the minor coat protein (pIII) of M13 phage. The displayed peptide 12-mers are expressed at the N-terminus of pIII. The library consists of about 1.9×109 electroporated sequences, amplified once to yield about 20 copies of each sequence in 10 μl of the supplied phage. Three biopannings were performed according to the instruction of the manufacturer with some modifications. Briefly, 10 μg of biotinylated mAb D32.10 were coupled to 35 mm polystyrene Petri dish (Falcon) coated with 40 μg of streptavidin. The dish was incubated overnight at 4° C. and washed six times with 50 mM Tris, 150 mM NaCl, pH 7.5 (TBS) containing 0.5% Tween-20 (TBS-T). In the first round of selection, 4×1010 phages from the initial library were allowed to react with the dish bound IgG for 4 h at 4° C. under shaking conditions. The unbound phages were removed by repetitive washes with TBS-T. The bound phages were then eluted from the dish with 400 μl of elution buffer (0.1 N HCl, pH adjusted to 2.2 with glycine, 1 mg/ml BSA). After neutralisation with 75 μl of 1 M Tris-HCl pH 9.1, the eluted phages were then amplified by infecting 20 ml of a 1:100 dilution of an overnight culture of E. coli ER2537 (recA+strain cells), as recommended in the instruction manual. The culture was incubated for 4.5 h at 37° C. with vigorous shaking The supernatants were obtained and precipitated with polyethyleneglycol (PEG) as previously described (Scott et al., 1990). In the second and third rounds of selection, 20% of the amplified phages from the preceding round were preincubated overnight at 4° C. with the biotinylated mAb D32.10 at the final concentration of 10 and 1 nM, respectively, before being added to the 35-mm polystyrene Petri dish coated with 10 μg of streptavidin. The procedure was then identical to the first round. The phages from the third biopanning eluate were then cloned and amplified for DNA sequencing and immunoanalysis. For DNA sequencing, single-stranded DNA was prepared from the purified phages as described by Sambrook et al. (1982). The nucleotide sequence of the gene III inserts was determined according to a modified version of the method of Sanger (Sanger et al., 1977) with an Applied Biosystems DNA sequencer (Model 377A) using the BigDye™ Terminator Cycle Sequencing Ready Reaction kit (Perkin-Elmer). Cycle sequencing was performed with a primer 5′ HO—CCCTCATAGTTAGCGTAACG-OH 3′ (SEQ ID NO: 4) corresponding to the pIII gene sequence. The aminoacid sequence of the insert was deduced from the nucleotide sequence.

For ELISA on supernatant phages, rows of ELISA plate wells were coated with 100 μl of either mAb D32.10 or an irrelevant mAb at the final concentration of 100 μg/ml in 0.1 M NaHCO3 buffer (pH 8.6). The plates were incubated overnight at 4° C. and then were blocked with 0.1 M NaHCO3 buffer (pH 8.6) containing 5 mg/ml of BSA. After a 2 hour incubation at 4° C., the plates were washed six times with TBS containing 0.5% Tween. Four fold serial dilutions of each phage clone were added to each well of the microtiter plate in a final volume of 100 μl of TBS-T, starting with 1012 virions in the first well of a row and ending with 2×105 virions in the 12th well. The plates were incubated for 2 h at room temperature with agitation and were then washed six times with TBS-T as above. The bound phages were detected in a sandwich assay using a horseradish peroxydase-conjugated anti-M13 mAb at a 1:5,000 dilution (Pharmacia). The plates were developed using a commercial color kit (bioMerieux) containing OPD and H2O2. After 10 min of incubation, the plates were read at 492 nm with an ELISA plate reader. For each phage clone dilution, the results were expressed as the difference between the value obtained with the tested anti-HCV mAb and the value obtained with the irrelevant mAb. The results were then confirmed by testing optimal dilutions of the immunoreactive clones in triplicate.

For sequence analysis, the amino acid sequences of peptides were compared to the HCV E1 and E2 protein sequences by use of the Mac Vector, Ver. 4.5 software (Kodak). Basically, the regions of highest similarity were detected with the LFASTA program, which tentatively searches for best local identities (Pearson and Lipman, 1988).

After the three rounds of selection, 4% of the phage input were found in the eluate indicating amplification of specifically bound phages. Thus, 88 clones were randomly isolated, their DNAs were sequenced and the amino acid sequences of inserts were deduced. Forty eight different sequences were obtained and some of them were found in several examples. However, when tested in an ELISA test for their immunoreactivity with D32.10, none of them gave a positive signal indicating that the binding affinity was too low to be detectable. The 48 clone sequences were compared to the sequences of HCV E1 and E2. Five and three sequences presented similarities with residues of E1 located in the 292-305 region and in the 347-356 region respectively (similarities are indicated in bold in Table 1), whereas 7, 4 and 2 sequences shared some similarities with residues of E2 located in the regions 481-501, 610-631 and 685-698 respectively (similarities are indicated in bold in Table 2).

TABLE 1
E1 (289-307)QLFTFSPRRHWTTTQGCNCS
(SEQ ID NO: 5)
Clone 1SPLRHYELPLIQ
(SEQ ID NO: 6)
Clone 2WPHNHSTHSRTH
(SEQ ID NO: 7)
Clone 3FPKYHPRFHKHA
(SEQ ID NO: 8)
Clone 4SQRSRHWHDVPK
(SEQ ID NO: 9)
Clone 5TSQPRWHQKPAT
(SEQ ID NO: 10)
E1 (343-363)AILDMIAGAHWGVLAGIAYFS
(SEQ ID NO: 11)
Clone 6WKMPRATDWNLR
(SEQ ID NO: 12)
Clone 7HWGNHSKSHPQR
(SEQ ID NO: 13)
Clone 8WHRTPSTLWGVI
(SEQ ID NO: 14)

TABLE 2
E2 (481-501)DQRPYCWHYPPKPCGIVPAKS
(SEQ ID NO: 15)
Clone 9WHKLPGHPRTV
(SEQ ID NO: 16)
Clone 4SQRSRHWHDVPK
(SEQ ID NO: 9)
Clone 10TFAWHKPRVNLG
(SEQ ID NO: 17)
Clone 11TSQPRWHQKPAT
(SEQ ID NO: 10)
Clone 12HSSWYIQHFPPL
(SEQ ID NO: 18)
Clone 13FPAHPLPRLPSL
(SEQ ID NO: 19)
Clone 8WHRTPSTLWGVI
(SEQ ID NO: 14)
E2 (610-631)DYPYRLWHYPCTINYTIFKIRM
(SEQ ID NO: 20)
Clone 1SPLRHYELPLIQ
(SEQ ID NO: 6)
Clone 14WHWNKPIIRPPLR
(SEQ ID NO: 21)
Clone 15QPYKLQAAATLY
(SEQ ID NO: 22)
Clone 6WKMPRATDWNLR
(SEQ ID NO: 12)
E2 (685-698)LSTGLIHLHQNIVD
(SEQ ID NO: 23)
Clone 16HLYHKNRNHHIAY
(SEQ ID NO: 24)
Clone 17WSPGQQRLHNST
(SEQ ID NO: 25)

b. Peptide Synthesis. In order to evaluate the significance of these different localizations on both E1 and E2 sequences, the regions 291-315 and 347-356 of E1 as well as the regions 473-498, 607-627 and 686-697 of E2 were reproduced as overlapping synthetic pentadecapeptides offset by one and tested for their immunoreactivity with D32.10. The simultaneous synthesis of different peptide sequences was performed on a nitrocellulose membrane using 9-fluorenylmethoxycarbonyl aminoacid chemistry (Frank, 1988)

Each peptide was generated in nanomolar quantities suitable for immunological detection. Antibody reactivity to membrane bound peptides was analyzed by an indirect colorimetric immunoassay as described previously (Jolivet-Reynaud, 1998). Spots corresponding to peptides with antibody reactivity produced a positive blue signal. Intensity of spots was estimated by visualization and expressed as relative intensity on a scale ranging from 0 to 5.

A strong positive signal was obtained with peptides corresponding to the 292-306 E1 region whereas the 347-356 E1 region was not recognized by D32.10. Peptides corresponding to the E2 regions 482-499 and 612-626 respectively were also immunoreactive with D32.10 and no signal was detected with the E2 region 686-697. The regions 292-306 of E1 as well as the regions 480-494, and 608-622 of E2 as pentadecapeptides interacted with D32.10 in ELISA.

Using overlapping octapeptides, the 297RHWTTQGCNC316 (SEQ ID NO: 1) region of the HCV E1 protein and both 613YRLWHYPCT621 (SEQ ID NO: 3) and 480PDQRPYCWHYPPKPC494 (SEQ ID NO: 2) regions of the HCV E2 protein were reactive with D32.10. The two regions identified in E2 contain the same motif WHYP (SEQ ID NO: 28) suggested by Yagnik et al. (2000) to be involved in the heterodimerization of E1E2. Indeed it is difficult to discriminate these regions, but as two non overlapping zones: 479GPDQRPYC486 (SEQ ID NO: 26) and 487WHYPPKPC494 (SEQ ID NO: 27) separately bound to D32.10, this suggests that D32.10 specifically recognizes each octapeptide, and so the complete sequence (480-494).

Example 2

Characterization and Purification of Serum Derived HCV Viral Particles

To separate the different HCV populations, the final HCV-enriched pellet was subjected to isopycnic centrifugation (210,000 g for 48 h at 4° C.) in a sucrose density gradient (10 to 60%, w/w). Fractions (0.6 ml) were collected, and the density of each was determined by refractometry. HCV-RNA content was analyzed by quantitative RT-PCR (Amplicor Monitor, Roche), HCV core protein content was measured with the Ortho-Clinical Diagnostics test and the HCV viral particles antigenic reactivity towards mAb D32.10 was measured by indirect EIA. Indirect EIA was carried out as indicated above except that the wells were coated with the different fractions diluted from 10−1 to 10−4.

Three different populations were identified (FIG. 4). One population (fractions 3 to 5) banded at a sucrose density of 1.06-1.08 g/ml and was substantially devoid of viral envelope as evidenced by the negative results obtained by indirect EIA with D32.10, but contained HCV RNA (about 2.105 UI/ml) and HCV core protein (from about 2 to 4 pg/ml). These seem to be non-enveloped viral particles. Another population (fractions 8 to 10 of figure) banded at a sucrose density of 1.14-1.15 g/ml and was substantially devoid of HCV RNA (less than about 104 to 105 UI/ml) and of HCV core protein (about 1 pg/ml) but contained high levels of particles responding to D32.10 (from about 1 to 3.8 OD45 nm units). These HCV subviral particles seem to contain only the HCV viral envelope. The third population (fractions 11 to 14) banded at a sucrose density of 1.20-1.21 g/ml and contained particles with high levels of HCV RNA (more than about 5.105 to 106 UI/ml) and of HCV core protein (from about 2.5 to 8 pg/ml) and responding to D32.10 (from about 0.5 to 1.5 OD450 nm units). Hence, this population contains substantially only purified HCV enveloped complete viral particles.

The viral particles contained in the HCV-enriched pellet (FIG. 5A), and in the second (FIG. 5C) and third populations (FIG. 5B) were immunoprecipitated by D32.10 and observed by electron microscopy. Several preparations of HCV viral particles have been analysed by this procedure. The HCV enveloped subviral particles appear as spherical particles with an average diameter of about 30 nm (33.08 nm) (FIG. 5C), whereas the HCV enveloped complete viral particles appear equally spherical but with an average diameter of about 50 nm (48.72 nm) (FIG. 5B).

Example 2

bis—Characterization and Purification of Serum Derived HCV Viral Particles

Another analysis of the population of HCV viral particles present in the plasma of infected patients was also carried out by the inventors.

Purification of Serum-Derived HCV Particles. The HCV enriched pellet obtained in Example 1 (HCV-L) was used for physico-chemical, immunological and morphological studies, as well as a pellet derived from the plasmapheresis of another patient (HCV-Fan, genotypela/2a), which showed chronic hepatitis C(CH-C) with type II cryoglobulinemia associated with severe cutaneous vascularitis, requiring regular plasma exchanges. As for the HCV-L patient, the latter HCV-Fan patient showed abnormal elevated serum aminotransferase (ALT/AST) levels, was positive for anti-HCV antibodies, and negative for all HBV and HIV markers.

Seven preparations were thus characterized. They were positive for HCV RNA from 106 UI/ml to 2-3×107 UI/ml (HCV-Fan, HCV-L) in the quantitative Amplicor HCV RNA Monitor test (Roche), corresponding to 2.5×106 copies/ml to 5-7.5×10 copies/ml.

To separate the different HCV populations, the final HCV-enriched pellet was subjected to isopycnic centrifugation (200,000 g for 48 h at 4° C.) in a sucrose density gradient (10 to 60% or 30 to 45%, w/w). Fractions (0.6 ml) were collected, and the density of each was determined by refractometry. HCV-RNA content was monitored by quantitative RT-PCR (Amplicor Monitor, Roche). HCV envelope (env) reactivity was detected using MAb D32.10 by indirect EIA, as described above, after coating of each fraction at different dilutions (1/10 to 1/10000) on solid phase. HCV core antigen was assayed by using quantitative Ortho assay and western blotting using a mixture of anti-core monoclonal antibodies (7G12A8, 2G9A7 and 19D9D6) (Jolivet-Reynaud et al., 1998; Menez et al., 2003).

Briefly, the measurement of total HCV core antigen in the HCV-enriched pellets and in each fraction collected from sucrose gradient was performed by using Ortho® trak-C™ assay (Ortho-Clinical Diagnostics, Inc). Ortho® trak-C™ assay is a quantitative immuno-capture assay that uses several monoclonal antibodies with specificity to different regions of the HCV core antigen. The procedure uses a pretreatment step to make sample preparation free, immune-complexed, and virion associated antigen available to assay (Aoyagi et al., 1999). HCV RNA was assessed by a quantitative RT-PCR developed by Roche Molecular System (Meylan, France), Amplicor™ HCV Monitor™. The amplification step involves a single tube, single enzyme (rTth DNA polymerase), single primer pair combined reverse transcription and DNA polymerisation (Colucci and Gutekunst, 1997). The detection was performed by a colorimetric microwell plate assay. Control of PCR carryover contamination included AmpErase™ which has been incorporated into the assay to inactive contaminating amplified DNA (Longo et al., 1990).

The HCV-enriched pellets were first subjected to an isopycnic centrifugation in sucrose density gradient (10% to 60%). The presence of enveloped viral particles exhibiting E1E2-D32.10 reactivity was investigated by the indirect EIA method described above (FIGS. 14(A) and (C)) and western blotting (FIG. 14(B) and (D)) using the anti-E1E2 MAb D32.10, the specificity of which was previously well-defined in Example 1. As shown in FIGS. 14(A) and (C), E1E2-D32.10 reactivity in EIA (fraction diluted at 1/10000) was recovered from the fraction 9 (density of 1.12 g/ml) to the fraction 16 (density of 1.23 g/ml), with a peak between 1.12 and 1.17 g/ml, and a “shoulder” from 1.18 to 1.23 g/ml. No or very low D32.10 reactivity (fraction diluted at 1/10) was detected in fractions corresponding to the low-density complexes (1.006 to 1.10 g/ml). Similar results were obtained with the isolate HCV-L (FIG. 14(A)) of genotype 1b and with the isolate HCV-Fan (FIG. 14(C)) of genotype 1a/2a. Some fractions (6, 8, 10, 12, 14) of the isolate HCV-L (FIG. 14(B)) and all the fractions (4 to 18) of the isolate HCV-Fan were subjected to SDS-PAGE and western blotting with MAb D32.10. E1E2-D32.10 reactivity clearly appeared from fractions 8 (density # 1.10 g/ml) to the bottom of gradient. Noteworthy, the strongest E1E2 reactivity was observed in fractions 11, 12 and 13 (FIG. 14(D)) corresponding to the densities of 1.17 to 1.20 g/ml. Higher molecular weight (HMW) bands could be detected in these fractions, probably corresponding to oligomeric forms of E1 and E2 envelope glycoproteins present on the surface of viral particles banding at such densities. Another strong reactivity against both E2 and E1 was observed in fraction 9 (density of 1.13 g/ml) corresponding to the peak in EIA (FIG. 14(C)). All serum-derived HCV preparations gave the same equilibrium banding profiles of E1E2-D32.10 reactivity in sucrose density gradients (10 to 60%).

To further characterize the enveloped HCV populations, the HCV-Fan pellet was subjected to isopycnic centrifugation in a 30 to 45% sucrose gradient of type 1 (2 ml of 30%, 3 ml of 35%, 3 ml of 40%, 2 ml of 45%). Under these experimental conditions, two peaks of E1E2-D32.10 reactivity could be clearly individualized, as shown in FIG. 15(A). Indirect EIA revealed a first narrow peak (Peak 1) at a density of about 1.15 g/ml, and a second wide large peak (Peak 2) at a density of about 1.18 g/ml to 1.22 g/ml. Particles from sucrose gradient (30-45%) peaks 1 (fraction 8) and 2 (fractions 12 and 13) were subjected individually to a second equilibrium centrifugation. Peak 1 was centrifuged in a 30 to 45% sucrose gradient of type 2 (3 ml of 30%, 3 ml of 35%, 2 ml of 40%, 2 ml of 45%) (FIG. 15(B)). Only one narrow E1E2 antigenic peak (Peak 1) migrated to a density of 1.13 to 1.14 g/ml (about 32% sucrose, fractions 5 and 6). Peak 2 was centrifuged in a 30 to 45% sucrose gradient of type 3 (2 ml of 30%, 2 ml of 35%, 3 ml of 40%, 3 ml of 45%) (FIG. 15(C)). A major peak (Peak 2) sedimented much faster, showing maximum E1E2 antigenicity at a density of about 1.18 g/ml (corresponding to 40% sucrose). The latter E1 E2 antigenic peak (Peak 2) appeared asymmetric, moved forward the lower densities, and was slightly overlapped with E1E2 population banding at a sucrose density of 1.14 g/ml (Peak 1), suggesting a greater heterogeneity than the former E1E2 antigenic peak (Peak 1) (FIGS. 15(B) and (C)).

To elucidate the differences in buoyant density between the two populations of E1E2 enveloped viral particles (Peak 1 and Peak 2), the fractions from 30 to 45% sucrose gradient of type 1 described above were tested for HCV core antigen by the Ortho HCV core assay (FIG. 9(A)) and by Western blotting (FIG. 15(B)).

Briefly, the polypeptide specificity was determined by immunoblotting using HCV samples as antigenic probes (1-5 μg/ml). SDS-PAGE on 12.5% gels was performed under reducing conditions (2% SDS+5% 2-ME). After protein transfer onto PVDF membranes, anti-E1E2 MAb D32.10 (2 to 5 μg/ml) or anti-core MAbs 7G12A8, 2G9A7 and 19D9D6 (5 μg/ml each) were used as primary antibodies, diluted in 50% NHS, and IgGs bound were then detected by incubation with HRPO-conjugated (Fab′)2 fragment of anti-mouse immunoglobulins (diluted 1/10,000; purchased from Immunotech), as second antibody. The blots were developed with an enhanced chemiluminescence (ECL Plus) system from Amersham.

Both experimental approaches showed that maximum HCV core antigen reactivity was recovered in the second E1E2 antigenic peak (Peak 2) at a density of approximately 1.17 to 1.19 g/ml (40% sucrose). By analysis in Western blotting (FIG. 9(B)), the HCV core protein of 25 kDa was identified in the initial material (HCV-Fan pellet) and fractions 8 to 13. Bands at 50, 75 and 150 kDa were also detected, which could correspond to oligomers of HCV core gene encoded-product. HCV RNA was analyzed by the quantitative RT-PCR Monitor test (Roche) in all the fractions from initial 10 to 60% sucrose gradient (FIG. 10). Two peaks of viral RNA occurred at a density of 1.055 to 1.075 g/ml, and at a density of 1.16 to 1.21 g/ml. Total titer of the latter peak was of 106 IU/ml, and corresponded to the second E1E2 antigenic peak (Peak 2). In contrast, fraction 9 (density of 1.13 g/ml), which corresponded to Peak 1, contained the lowest titer of HCV RNA. Thus, peak 2 (approximately 1.17 to 1.20 g/ml) contained E1E2 complexes, HCV core protein, high titer of HCV RNA and is likely to represent complete virions. Peak 1 (1.13 to 1.15 g/ml) contained mainly E1E2 complexes and is likely to represent subviral particles (SVPs). The low density complexes (1.006 to 1.10 g/ml) contained only core and HCV RNA, corresponding to naked nucleocapsids.

Thus, using the D32.10 monoclonal antibody of the invention, the most prevalent types of enveloped viral particles found in the serum of chronic hepatitis C patients have been analyzed. By sedimentation in sucrose gradients of concentrated viral material, analysis of the protein composition (core and envelope proteins) by immunoassays and immunoblotting, coupled with monitoring of HCV RNA by quantitative RT-PCR, two populations of E1 E2 enveloped particles which sedimented at two distinct densities of 1.13-1.15 g/ml and 1.17-1.21 g/ml, were identified respectively. The first population corresponded to subviral particles (SVPs) containing the E1 and E2 envelope proteins, but lacking nucleocapsids. The second population could correspond to HCV virions, since these particles contained the E1 and E2 envelope proteins, the core proteins and high titer of HCV RNA.

Morphological Characterization of Serum-Derived HCV Particles. Electron microscopy of HCV-Fan pellet after immunoprecipitation by E1E2(D32.10)-specific monoclonal antibody (immuno-electron microscopy or IEM), was performed according to the following procedure.

Briefly, serum-derived HCV preparations (2, 20 or 200 μg) or fractions from the gradients (0.1 ml) were mixed with 0.1 ml of MAb D32.10 (5 μg) or irrelevant monoclonal antibody (F35.25, HBV anti-preS1) (Petit et al., 1989) of the same isotype (IgG1). Incubation was performed at 37° C. for 1 h, then at 4° C. for 16 h. The mixture was then diluted in 10 ml of TNE buffer and centrifuged at 48,000 g for 1 h. The pellet was resuspended in 0.1 ml of TNE buffer. D32.10-immunoprecipitated HCV particles were absorbed for 5 min onto glow-discharged Formvar/carbon-coated cupron grids and stained for 4 min with 1% uranyl acetate (pH 4.5). The preparations were then visualized using a JEOL 100 CX electron microscope (Imaging facility, Laennec Faculty, Lyon, France). For indirect immuno-gold labeling, the grids were incubated with a 1:50 dilution of goat anti-mouse IgG-colloidal gold particles (10-nm diameter; BioCell Research Laboratories), as second antibody.

Control experiments showed that the size heterogeneity of preparations varied with the antigen:antibody ratio (results not shown). In order to preserve morphological integrity, isolation procedure of HCV particles involved only precipitation and sucrose gradient centrifugation. Under such experimental conditions, a relatively well-defined particle size distribution pattern was obtained, as shown in FIG. 11(A). The asymmetric peak centered at about 35 nm Particles of this size (30 to 40 nm) were predominant (56%). Essentially the majority of particles (80%) had a diameter greater than 30 nm, with a mean value of 38.28 nm (125 out of 156 particles), 20% of them between 40 to 50 nm (mean value=43.61 nm) and 4% between 50 and 60 nm (mean value=55.69 nm). Only 20% of all particles had a diameter smaller than 30 nm (mean value=26.35 nm). Electron micrographs (FIG. 11(B)) showed that HCV particles are characterized by a relatively regular smooth surface. The panels a and b of FIG. 11(B), illustrate the heterogeneity in size of HCV enveloped particles. FIG. 11(B), panel c shows particles with a diameter of 50 nm, and panel d those with a diameter of 35 nm A quite homogenous higher density appeared on the surface of particles of 35 nm (FIG. 11(B), panel d), whereas a lighter area of lower density could be clearly observed in the center of particles with a diameter of 50 nm (FIG. 11(B), panel c). No particles were visualized by IEM when using an irrelevant primary monoclonal antibody (FIG. 11(B), panel e). Particles from sucrose gradient peaks 1 and 2 were immunoprecipitated, stained, and also analyzed by IEM (FIG. 12(A)-(B)). As shown in FIG. 12(A), the predominant species (85.4%) in peak 1 was a spherical particle with a diameter of about 20 nm (35 out of 41 particles, mean value=20.6 nm). Only 14.6% of particles had a diameter greater than 30 nm, probably corresponding to an overlap with peak 2. Peak 2 appeared somewhat more heterogeneous in size than the peak 1 material (FIG. 12(B)). However, the most prevalent forms (77.5%) in this population had a mean diameter of about 41 nm (69 out of 89 particles, mean value=41.15 nm), which could correspond to the diameter of the whole HCV virion. A few of the smaller 30-nm-diameter particles (13.5%) were visible in this preparation, and probably result from an overlap with peak 1. The heterogeneous major faster-sedimenting peak (Peak 2) consisted primarily of 35 and 50-nm-diameter large particles, whereas the homogeneous minor slower-sedimenting peak (Peak 1) consisted of approximately 20-nm-diameter small particles. Taken together, the morphological studies indicate that two size classes of E1E2 enveloped HCV particles co-exist in the serum of infected patients. By indirect immuno-gold labeling (FIG. 13), all spherical enveloped particles, previously identified by IEM, specifically fixed 10-nm-gold-labeled secondary antibody.

Thus, by immuno-electron microscopy with MAb D32.10, a relatively well-defined particle size distribution of the total enveloped population (HCV-enriched pellet) and of each E1 E2 population separately, was determined. The enveloped HCV particles are heterogenous in size, but two distinct size classes of particles could be readily identified. A predominent species consisted of large particles with a diameter which varied from 35 nm to 50 nm, likely depending on the lattice structure of the envelope, corresponding to complete capsid-containing enveloped particles. Another more homogenous species consisted of small particles with a diameter of about 20 nm, corresponding to capsidless SVPs. All these particles which bound to MAb D32.10 were further identified and visualized by electron microscopy using an indirect labelling with anti-mouse IgG-gold particles.

Formation of noninfectious SVPs, in addition to infectious virions, is a common characteristic of flavivirus infections (Russel et al., 1980). It has been suggested that these represent capsidless empty viral envelopes (Mason et al., 1991). It also occurs in cells infected with hepatitis B virus (HBV), and secreted SVPs are produced by expression of envelope proteins alone (Laub et al., 1983). These particles are smaller than virions. This demontrates that these proteins are intrinsically capable of self-forming specific particulate structures in the presence or absence of a core. These particles are assembled and undergo the same maturation process as the whole virions, including glycosylation and carbohydrate processing. Immunization with such recombinant envelope SVPs shows them to be excellent protective immunogens in animal models (Konishi et al., 1992; Heinz et al., 1995) as well as in humans (Leroux-Roels et al., 1994).

The reactivity of complete virions and SVPs with MAb D32.10 shows that El(297-306)-E2(480-494)(613-621) conformational epitope is presented in essentially the same way on the surfaces of HCV virions and SVPs. However, such epitope detected on virions and SVPs derived from serum is lacking on particles obtained by genetic recombination and produced in heterologous systems: baculovirus/insect cells (Baumert et al., 1999) or retrovirus/293T cells (Bartosch et al., 2003), suggesting that this composite site is either absent and/or present on separate inaccessible regions on the surfaces of these recombinant HCV particles. The former HCV-like particles (HCV-LPs) were retained in the ER, and only a small fraction of the latter HCV pseudo-particles (HCVpp) reached the cell surface. It is worth noting that MAb D32.10 is not reactive with either HSA or with γ- or μ-chains of immunoglobulins. It is able to specifically immunoprecipitate E1, E2, as well as E1E2 heterodimers, when these two proteins are expressed separately or together by vaccinia virus recombinants in HepG2 cells. It thus reacts mostly with disulfide-bond linked-aggregates and more faintly with E1E2 non covalently-linked mature complex. However, it does not recognize denatured recombinant E1 or E2 proteins expressed in such a heterologous system by blotting analysis. This is in favour of an arrangement of E1, E2 and E1E2 on the serum-derived virus surface which is different from the one found in recombinant HCV particles. The natural structure depends on assembly conditions. If the conditions that regulate formation of a unique structure are absent in heterologous systems, the dimer arrangement in the VLPs and HCVpp is not similar, contrasting with the similarity of SVPs and virion surfaces, when both are isolated from the serum of infected patients. One particularly noteworthy feature of the packing of the envelope proteins (E) of flaviviruses, including tick-born encephalitis virus (TBEV), in recombinant subviral particles (RSPs) and virions, is that they lie flat on the viral surface, in a head-to-tail orientation (Ferlengi et al., 2001). The lateral surface on domain III, which has an Ig-like fold and is implicated in receptor binding, is accessible, while the fusion peptide is an internal loop (Rey et al., 1995). So, flaviviruses enter cells by receptor-mediated endocytosis and low-pH-induced fusion in endosomes (Rice, 1996). An assembling particle is believed to acquire its envelope by budding through the membrane of the ER or an intermediate compartment of the early secretory pathway. The particles are then transported through the trans-Golgi network (TGN) to the plasma membrane, mature and so acquire complex sugars. All these steps are crucial for virus assembly and play a critical role in many phases of the replication cycle.

Unexpectedly, all HCV particles seemed to exhibit similar biophysical properties whatever their origin. The sucrose gradient sedimentation pattern of HCV-LPs assembled in insect cells, derived from strain HCV-J cDNA or from infectious clone H77c, was 1.14 to 1.20 g/ml in sucrose equilibrium gradients (WelLnitz et al., 2002). The majority of particles had a diameter of 40 to 60 nm However, all epitopes presented on the E1 and E2 ectodomains of the outer surface of the HCV-LPs (WelLnitz et al., 2002) are distinct from the E1 and E2 specificity of MAb D32.10. So, even if the particles sediment at the same density in sucrose, they possess different immunoreactivities, which may be linked to an improper transport and/or folding, and obviously will result in different functional properties, in terms of cell binding and infectivity.

Contrary to serum enveloped HCV particles, HCV core particles isolated from the plasma of HCV-infected individuals seem to be similar to nucleocapsid-like particles produced in insect cells. HCV nucleocapsids banded at a density of 1.32 to 1.34 g/ml in the CsCl gradient and were very heterogenous in size, 38 to 62 nm in diameter (Maillard et al., 2001). All these particles were reactive with anti-core MAbs, but not with anti-E1 and anti-E2 MAbs produced by immunization with recombinant proteins (Deleersnyder et al., 1997; Dubuisson et al., 1994).

So, different types of HCV-related particles (HCV-LPs, serum HCV nucleocapsids, serum HCV virions) possess some similarities in size and/or in density, but exhibit different antigenic properties (improper or proper folding of E1E2 complexes, enveloped virions or nonenveloped nucleocapsids). Therefore, altogether this demontrates that it is crucial to have good immunological tools for analyzing the true antigenic properties of HCV particles, and so for identifying the natural HCV virions.

In the light of these data and those of others (Andre et al., 2002; Maillard et al., 2001), a better knowledge of complete spectrum of different forms of HCV-related particles circulating in the serum of chronic hepatitis C patients could be acquired. Low-density fractions in sucrose gradient (1.06 g/ml) contained capsid-like structures, associated with lipoproteins to form lipo-viro-particles (LVP), and with HCV RNA and immunoglobulins depending on individual variations (Andre et al., 2002). LVP appeared as large particles of more than 100 nm in-diameter, and bound hepatocyte cell line through the LDL receptor (Andre et al., 2002). Nonenveloped HCV nucleocapsids isolated from detergent treated sera had a buoyant density of 1.32 to 1.34 g/ml in CsCl, were heterogeneous in size (Maillard et al., 2001), and were recently shown to exhibit F7R-like activity (Maillard et al., 2004), which could explain the association of HCV RNA-containing particles with “non-immune” antibodies as HCV core-IgG complexes as high-density (1.28 to 1.35 g/ml) in sucrose gradient. The fractions of intermediate density, between 1.13 to 1.21 g/ml, were identified here as E1E2 enveloped HCV particles. The average density of the presumptive entire HCV virion (1.18 to 1.20 g/ml), consisting of a nucleocapsid, lipid membrane, and two E1 and E2 envelope glycoproteins, is similar to that of the flaviviruses (Bradley et al., 1991). The hypothesis of lateral envelope protein interactions, deduced from the fine specificity of MAb D32.10, is also similar to organization of the envelope of recombinant SVPs from TBEV and whole virus particles (Schalich et al., 1996). Finally, the presence in serum of HCV-infected patients of SVPs containing only the E1E2 envelope proteins, showing different sedimentation properties (1.13 to 1.15 g/ml) and different particle sizes (a 20 nm) but similar antigenic characteristics as whole virus particles, supports some similarities between HCV and the flaviviruses.

In conclusion, by using a new MAb D32.10, were identified for the first time: (i) HCV complete virions, containing both HCV RNA and core antigen, and expressing E1E2 envelope complexes on their surface, as “large particles”, with a diameter of 35-50 nm and banding at 1.17-1.21 g/ml; (ii) an HCV population of envelope SVPs, as “small particles”, devoid of capsid and HCV RNA with a diameter of 20-30 nm and banding at 1.13-1.15 g/ml.

Such enveloped particles may therefore represent a relevant model for investigating HCV envelope glycoprotein structure-function relationship, and an excellent candidate for vaccination approaches. In addition, it will be interesting to follow the spectrum of circulating HCV particles up in different groups of patients, and at different stages of the HCV-related disease.

Example 3

Monoclonal Antibody Directed Against Purified HCV Enveloped Complete Viral Particles

The fraction of the HCV-enriched pellet described above that was subjected to isopycnic centrifugation in a sucrose density gradient and containing the purified HCV enveloped complete viral particles, was used to prepare a monoclonal antibody. This fraction was used to immunize mice and isolate hybridomas as explained above. Thus, one monoclonal antibody, (D4.12.9) was obtained, which was shown to specifically recognize the natural HCV E2 protein in Western blotting experiments (FIG. 6) and the natural HCV viral particles evidenced in Example 2 (FIG. 7). Indeed, FIG. 7 shows that D4.12.9 recognizes both the HCV enveloped subviral particles (fractions 8 to 11) and the HCV enveloped complete viral particles (fractions 11 to 14), in a manner similar to D32.10.

Example 4

Epitopic Characterization of Anti-HCV Antibodies Derived from Infected Patients

The immunoreactivity of the E1 and E2 derived peptidic sequences reactive with D32.10 (Example 1) towards sera of HCV-infected patients was assessed by using peptides encompassing them. E1 (amino acids 292-306) and E2 (amino acids 480-494 and 608-622) sequences were produced as biotinylated synthetic peptides to be tested by ELISA with 11 sera from healthy individuals and 44 sera from HCV-infected patients (numbered A1 to A44).

The wells of microtitration plates were coated overnight at 4° C. with 100 μl of streptavidin at the concentration of 10 μg/ml in 0.1 M carbonate buffer (pH 9.6) and blocked for 1 h at 37° C. with PBS containing 10% goat serum. The plates were then washed three times with PBS containing 0.05% Tween-20 before adding 100 μl of a biotinylated peptide solution (10 μg/ml in PBS) for 2 h at 37° C. After a new wash with PBS-Tween, 100 PI of the tested serum diluted 1:50 in PBS-Tween containing 10% goat serum was added and incubated for 2 h at 37° C. The plates were washed again with PBS-Tween. The secondary antibody, peroxydase-conjugated goat anti-human IgG (Jackson ImmunoResearch Laboratories), was then added at a 1:5000 dilution in PBS-Tween-goat serum. The plates were incubated 1 h at 37° C. and then washed once more with PBS-Tween. The plates were developed using the Biomerieux color kit containing o-phenylenediamine and hydrogen peroxide. After 10 min of incubation, the plates were read at 492 nm with an ELISA plate reader. The reported values are the mean OD of triplicate.

A cut off recognition was calculated for each peptide (mean of the values obtained with HCV negative sera+3 standard deviations). It allowed to evidence positive responses with 6 out of 44 HCV-positive sera against E1 (amino acids 290-317) (FIG. 8(A)), 6 out of 44 against E2 (amino acids 471-500) (FIG. 8(B)) and 16 out of 44 against E2 (amino acids 605-629) (FIG. 8(C)). Sera A7, A14, A21, A33, A39 and A40 gave a positive signal with the three peptides whereas E2 (605-629) was also recognized by 10 more sera.

The presence in the sera of HCV-infected patients of specific antibodies able to react simultaneously with the three regions of E1 and E2 recognized by D32.10 mAb strongly supports their juxtaposition at the surface of circulating enveloped HCV viral particles and their immunogenicity in mice as well as in humans.

Example 5

Sequencing of the Fab fragment of the D32.10 Monoclonal Antibody Materials and Methods

Production of the D32-10 Fab. Secreted IgGs (isotype IgG1, κ) were purified from a D32.10 hybridoma culture by precipitation with 75% ammonium sulfate at pH 7.4, followed by affinity chromatography on a Protein A-Sepharose column The IgG obtained was treated with papain and purified again on a Protein A-Sepharose column to remove undigested immunoglobulins and Fc fragments and then by gel filtration on Sepharex 75. The purity was assessed by SDS-PAGE, protein A agarose and gel filtration. The resulting protein was loaded on 12% SDS PAGE and transferred to a PVDF membrane.

N-terminal Sequencing. N-terminal sequencing of the heavy and light bands on the membrane was performed by standard Edman degradation using a model 492 sequencing system (Applied Biosystems) followed by identification using a model 140C HPLC (Applied Biosystems) and analysis using the Model 610A (V2.1) software (Applied Biosystems). The following protein sequences were found: (i) For the light chain: N-I-V-L-T Q-S-P (ii) for the heavy chain: E-V-Q-L-Q-Q-S-G.

Bioinformatics Analysis and Primers Design. These protein sequences were compared to the whole murine repertoire of variable domains available at www.imgt.org. The cDNA sequences of all the variable domains starting with these protein sequences were aligned and compared, and oligonucleotide primers matching this alignment were designed. The alignment showed only two different cDNA sequences for each chain, so two oligonucleotides were designed for each chain:

for the first light chain (L1):
(SEQ ID NO: 29)
gaattcggatccAAC ATT GTG CTG ACC CAA TCT CCA;
for the second light chain (L2):
(SEQ ID NO: 30)
gaattcggatccAAC ATC GTG CTC ACT CAG TCT CCA;
for the first heavy chain (H1):
(SEQ ID NO: 31)
gaattcggatccGAG GTT CAG CTG CAG CAG TCT GGG;
and
for the second heavy chain (H2):
(SEQ ID NO: 32)
gaattcggatccGAG GTC CAG CTT CAG CAG TCT GGA,

wherein the sections in lower case letters correspond to restriction enzyme sites introduced in the oligonucleotides for cloning purposes, and the sections in capital letters correspond to the cDNA derived sequences corresponding to the protein sequence determined by N-terminal sequencing. In addition to these forward primers, a common reverse primer based on a polyT sequence was also design, including enzyme sequences and ordered (polyT30ctcgagaagcttTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT—SEQ ID NO: 33).

RNA Isolation, cDNA Amplification and Sequencing. Total RNA was extracted from fresh hybridoma cells using the RNeasy mini kit (Qiagen) and cDNA was generated by reverse transcription using SuperScript® III First-Strand Synthesis SuperMix kit (Invitrogen) and the polyT primer. The cDNA was amplified by PCR using the specific and polyT primers described above and the Phusion High-Fidelity PCR master mix (Finnzymes). All kits were used following the manufacturer recommendations. The PCR was performed on a MasterCycler (Eppendorf) with the following steps: 15 cycles of: 98° C. for 15 seconds/60° C. for 30 seconds (−1° C./cycle, “touchdown PCR”)/72° C. for 2 minutes followed by 30 cycles of 98° C. for 15 seconds/55° C. for 30 seconds/72° C. for 2 minutes.

Only the H1 and L1 primers gave a band after PCR amplification. The resulting PCR products were purified using the QIAquick PCR purification kit (Qiagent) and sequenced by Eurofins MWG biotech.

3-Dimensional Structure of the Fab Fragment. The immunoglobulin was purified from culture supernatants of hybridoma D32.10. Crystallization was performed at 3.5 mg/ml in 100 mM Hepes pH 7.5, 20% (w/v) PEG 4000 and 7% isopropanol. The crystals, which appeared after 3 days at room temperature, were frozen in liquid nitrogen in the same buffer containing 20% glycerol, and data were collected at the ESRF (Grenoble, France) on the ID29 beamline.

Results

Sequence of the D32.10 Heavy Chain. The sequence of the full length heavy chain H is presented below (SEQ ID NO: 34):

EVQLQQSGAELVKPGASVKLSCTPSGFNIKDTYIHWVKQRPEQGLEWIGM
INPANGNSNYDPKFQDKATITADTSSNTAYLQLSSLTSEDTAVYYCAEIT
TDFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFP
EPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNV
AHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTP
KVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELP
IMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTTGRPKAPQVYTIPPPKEQ
MAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVY
SKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

wherein the following domains have been identified:
  • a heavy chain variable region (SEQ ID NO: 35)

EVQLQQSGAELVKPGASVKLSCTPSGFNIKDTYIHWVKQRPEQGLEWIGM
INPANGNSNYDPKFQDKATITADTSSNTAYLQLSSLTSEDTAVYYCAEIT
TDFDVWGAGTTVTVSS

comprising:
  • IGHV14-3*02 (Immunoglobulin heavy variable 14-3*02-SEQ ID NO: 36):

EVQLQQSGAELVKPGASVKLSCTPSGFNIKDTYIHWVKQRPEQGLEWIG
MINPANGNSNYDPKFQDKATITADTSSNTAYLQLSSLTSEDTAVYYC
AEITTD

and a linker (SEQ ID NO: 37):

FDVWGAGTTVTVSS

and a heavy chain constant region region comprising:
  • IGHG1*02 (Immunoglobulin heavy constant gamma 1*02-SEQ ID NO: 38):

AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSG
VHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIV

linker (SEQ ID NO: 39):

PRDCGCKPCICT
  • IGHG1*01 (Immunoglobulin heavy constant gamma 1*01-SEQ ID NO: 40):

VPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDV
EVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNG
  • linker (SEQ ID NO: 41):

KEFKCRVNSAAFPAPIEKTISKTT
  • and IGHG1*02 (Immunoglobulin heavy constant gamma 1*02-SEQ ID NO: 42):

GRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAE
NYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTE
KSLSHSPGK

wherein the sequences of the CDRs (complementary determining regions), as underlined above, are as follows:

GFNIKDTYIH(SEQ ID NO: 43)
MINPANGNSN(SEQ ID NO: 44)
AEITTDFDVW(SEQ ID NO: 45)

Sequence of the D32.10 Light Chain. The sequence of the full length light chain L is presented below (SEQ ID NO: 46):

NIVLTQSPPSLAVSLGQRATISCRASESVDSYGNSFLHWYQQKSGQPPK
LLIYLASNLESGVPARFSGSGSRTDFTLTIDPLEADDAATYYCQQNNEA
PFTEGSGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKD
INVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSY
TCEATHKTSTSPIVKSFNRNEC

wherein the following domains have been identified:
  • a light chain variable region (SEQ ID NO: 47)

NIVLTQSPPSLAVSLGQRATISCRASESVDSYGNSFLHWYQQKSGQPPK
LLIYLASNLESGVPARFSGSGSRTDFTLTIDPLEADDAATYYCQQNNEA
PFTFGSGTKLEIK

comprising:
  • IGKV3-10*01 (Immunoglobulin kappa variable 3-10*01-SEQ ID NO: 48):

NIVLTQSPPSLAVSLGQRATISCRASESVDSYGNSFLHWYQQKSGQPPK
LLIYLASNLESGVPARFSGSGSRTDFTLTIDPLEADDAATYYCQQNNEAP
  • and a linker (SEQ ID NO: 49):
  • FTFGSGTKLEIK
  • and a light chain constant region: IGKC*01 (Immunoglobulin kappa constant *01-SEQ

ID NO: 50):

RADAAPTVSIFPPSSEQLTSGGASVVCFLNNEYPKDINVKWKIDGSERQ
NGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPI
VKSFNRNEC

wherein the sequences of the CDRs (complementary determining regions), as underlined above, are as follows:

ESVDSYGNSF(SEQ ID NO: 51)
YLASN(SEQ ID NO: 52)
QQNNEAPFT.(SEQ ID NO: 53)

Sequence of the DNA sequence of the D32.10 Heavy Chain. The DNA sequence of the full heavy chain H is presented below (SEQ ID NO: 54):

GAGGTTCAGCTGCAGCAGTCTGGGGCAGAACTTGTGAAGCCAGGGGCCT
CAGTCAAGTTGTCCTGCACACCTTCTGGCTTCAACATTAAAGACACCTA
TATACACTGGGTGAAACAGAGGCCTGAACAGGGCCTGGAGTGGATTGGA
ATGATTAATCCTGCGAATGGTAATTCTAATTATGACCCGAAGTTCCAGG
ACAAGGCCACTATAACAGCAGACACATCCTCCAACACAGCCTACCTGCA
ACTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTGCTGAG
ATTACGACGGACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCT
CCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATC
TGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGC
TATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCA
GCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCT
GAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTC
ACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAA
TTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGA
AGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACC
ATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGG
ATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCA
CACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGC
TCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGG
AGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAA
AACCATCTCCAAAACCACAGGCAGACCGAAGGCTCCACAGGTGTACACC
ATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCT
GCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTG
GAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGAC
ACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCA
ACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCT
GCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAATGA

Sequence of the DNA sequence of the D32.10 Light Chain. The DNA sequence of the full light chain L is presented below (SEQ ID NO: 55):

AACATTGTGCTGACCCAATCTCCACCTTCTTTGGCTGTGTCTCTAGGGC
AGAGGGCCACCATATCCTGCAGAGCCAGTGAAAGTGTTGATAGTTATGG
CAATAGTTTTTTGCACTGGTACCAGCAGAAATCAGGACAGCCACCCAAA
CTCCTCATCTATCTTGCATCCAACTTAGAATCCGGGGTCCCTGCCAGGT
TCAGTGGCAGTGGGTCTAGGACAGACTTCACCCTCACCATTGATCCTTT
GGAGGCTGATGATGCTGCAACCTATTACTGTCAGCAAAATAATGAGGCT
CCATTCACGTTCGGCTCGGGGACAAAGTTGGAGATAAAACGGGCTGATG
CTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATC
TGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGAC
ATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCC
TGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAG
CAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTAT
ACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCT
TCAACAGGAATGAGTGTTAG

Additional results. The crystals of the Fab fragment of the D32.10 monoclonal antibody, obtained as described above, were found to diffract to 2.9 A and belong to the space group P212121. The structure of Fab D32.10 was obtained by molecular replacement using pdb ref 1IQW as a search model. After completion of the refinement of the atomic parameters, the R and Rfree values were 0.23 and 0.30, respectively. Finally, the complete Fab with VH and VL of D32.10 has been successfully traced. Sequence analysis of D32.10, cloning, expression and characterization of its single-chain FIT (scFv) construction, and co-crystallization of Fab D32.10 with peptides containing the minimal epitope were performed to understand the nature of the interaction of D32.10 with the HCV E1E2 envelope antigen.

Other Embodiments

Other embodiments of the invention will be apparent to those skilled in the art from a consideration of the specification or practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope of the invention being indicated by the following claims.

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