Prevention and treatment of HCV infection employing antibodies directed against conformational epitopes Number:7,091,324 from the United States Patent and Trademark Office (PTO) owispatent

Title: Prevention and treatment of HCV infection employing antibodies directed against conformational epitopes

Abstract: Conformational epitopes of the envelope protein E2 of the Hepatitis C virus (HCV) have been identified and characterized using a panel of monoclonal antibodies derived from patients infected with HCV. These conformational epitopes have been determined to be important in the immune response of humans to HCV and may be particularly important in neutralizing the virus. Based on the identification of these conformational epitopes, vaccines containing peptides and mimotopes with these conformational epitopes intact may be prepared and administered to patients to prevent and/or treat HCV infection. The identification of four distinct groups of monoclonal antibodies with each directed to a particular epitope of E2 may be used to stratify patients based on their response to HCV and may be used to determine a proper treatment regimen.

Patent Number: 7,091,324 Issued on 08/15/2006 to Foung,   et al.

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Inventors:	Foung; Steven K. H. (Stanford, CA), Hadlock; Kenneth G. (San Francisco, CA), Keck; Zhen-yong (Redwood City, CA)
Assignee:	Board of Trustees of Leland Stanford Junior University (Stanford, CA)
Appl. No.:	09/728,720
Filed:	December 1, 2000

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Related U.S. Patent Documents

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	Application Number	Filing Date	Patent Number	Issue Date
	09430489	Oct., 1999	6692908
	09187057	Nov., 1998

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Current U.S. Class:	530/388.3 ; 530/387.3; 530/388.15; 530/389.4
Current International Class:	C07K 16/10 (20060101)
Field of Search:	530/388.3,388.1,388.15,387.3,389.4 435/5,339 424/142.1,149.1,133.1,141.1,147.1,159.1

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Foreign Patent Documents

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Other References

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Landford, et al., "Analysis of Hepatitis C Virus Capsid, E1, and E2/NS1 Proteins Expressed in Insect Cells", Virology, 197: 225-235, 1993. cited by other . Mahaney, et al., "Genotypic Analysis of Hepatitis C Virus in American Patients", Hepatology, 20: 1405-1411, 1994. cited by other . Meola, et al., Derivation of Vaccines from Mimotopes, Immunologic Properties of Human Hepatitis B Virus Surface Antigen Mimotopes Displayed on Filamentous Phage, J. Immunol. 154:3162-3172, 1995. cited by other . Mondelli, et al., "Significance of the Immune Response to a Major, Conformational B-Cell Epitope on the Hepatitis C Virus NS3 Region Defined by a Human Monoclonal Antibody", J. Virol. 68:4829-4836, 1994. cited by other . Moradpour, et al., "Characterization of Three Novel Monoclonal Antibodies Against Hepatitis C Virus Core Protein", J. Med. Virol. 48:234-241, 1996. cited by other . 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Primary Examiner: Mosher; Mary E.
Attorney, Agent or Firm: Choate, Hall & Stewart LLP Jarrell; Brenda Hershbach Baker; C. Hunter

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Government Interests

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GOVERNMENT LICENSE RIGHTS

The U.S. government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to license others on reasonable terms as provided for by the terms of grant no. DA60596 and HL33811 awarded by the National Institutes of Health (NIH).

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Parent Case Text

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CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of patent application U.S. Ser. No. 09/430,489, filed Oct. 29, 1999, now U.S. Pat. No. 6,692,908, which is a continuation-in-part of patent application U.S. Ser. No. 09/187,057, filed Nov. 5, 1998 abandoned. Each if these applications is incorporated herein by reference.

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Claims

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What is claimed is:

1. An isolated antibody that binds to a conformational epitope of a Hepatitis C virus E2 protein, wherein the epitope is found in Hepatitis C virus of more than one genotype, and wherein the antibody is selected from the group consisting of CBH-2, CBH-4G, CBH-5, CBH-7, CBH-8C, and CBH-11, or binds to the same conformational epitope as that bound by an antibody selected from the group consisting of CBH-2, CBH-4G, CBH-5, CBH-7, CBH-8C, and CBH-11.

2. An isolated antibody that binds to a conformational epitope within amino acids 411 through 644 of E2 protein of Hepatitis C virus 1b, wherein the antibody-binds to the E2 protein of Hepatitis C virus of more than one genotype, wherein the antibody is selected from the group consisting of CBH-2, CBH-5, CBH-8C, and CBH-11, or binds to the same conformational epitope as that bound by an antibody selected from the group consisting of CBH-2, CBH-5, CBH-8C, and CBH-11.

3. An isolated antibody that binds to a conformational epitope within amino acids 470 through 644 of E2 protein of Hepatitis C virus 1b, wherein the antibody is capable of binding to the E2 protein of Hepatitis C virus of more than one genotype, wherein the antibody is CBH-4G or CBH-7, or binds to the same conformational epitope as that bound by CBH-4G or CBH-7.

4. An isolated antibody that binds to the epitope recognized by CBH-2, -4D, -4B, -4G, -5, -7, -8C or -11.

5. The isolated antibody of claim 1 wherein the antibody inhibits binding of HCV E2 protein to CD81.

6. A cell line expressing the isolated antibody of claim 1.

7. The cell line of claim 6 wherein the cell line is a B cell line.

8. The cell line of claim 6 wherein the cell line is a human cell line.

9. The cell line of claim 6 wherein the cell line is a mammalian cell line.

10. The cell line of claim 6 wherein the cell line is a eukaryotic cell line.

11. The cell line of claim 6 wherein the cell line is a hybridoma.

12. The cell line of claim 6 wherein the cell line has been transformed with Epstein-Barr virus (EBV).

13. The cell line of claim 6 wherein the cell line has been infected with a virus.

14. The isolated antibody of claim 1, 2, 3, or 4, wherein the antibody is a monoclonal antibody.

15. The isolated antibody of claim 1, 2, 3, or 4, wherein the antibody is a human antibody.

16. The isolated antibody of claim 1, 2, 3, or 4, wherein the antibody is a humanized antibody.

17. The isolated antibody of claim 1 wherein the antibody is a mammalian antibody.

18. A combination of two or more isolated antibodies wherein at least two of the antibodies bind to different conformational epitopes of E2 protein of Hepatitis C virus of more than one genotype, wherein each antibody is selected from the group consisting of CBH-2, CBH-4G, CBH-5, CBH-7, CBH-8C, and CBH-11, or binds to the same conformational epitope as that bound by an antibody selected from the group consisting of CBH-2, CBH-4G, CBH-5, CBH-7, CBH-8C, and CBH-11.

19. The combination of claim 18, wherein the combination shows increased total binding of the combined antibodies to E2 protein of Hepatitis C virus compared to the binding shown by any of the antibodies individually.

20. The combination of claim 18 comprising CBH-7 and CBH-4G.

21. The combination of claim 18 comprising CBH-7 and CBH-17.

22. The combination of claim 18 comprising CBH-7 and CBH-5.

23. The combination of claim 18 comprising CBH-7 and CBH-2.

24. The combination of claim 19, wherein each antibody in the combination binds to a different epitope.

25. An isolated antibody that binds to a conformational epitope of Hepatitis C virus E2 protein, wherein the antibody is selected from the group consisting of CBH-4B and CBH-4D.

26. A combination of two or more isolated antibodies, at least two of which bind to different conformational epitopes of E2 protein of Hepatitis C virus, wherein each antibody is selected from the group consisting of CBH-4B, CBH-4D, CBH-4G, and CBH-7.

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Description

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INTRODUCTION

1. Technical Field

The field of this invention is related to the preparation of human monoclonal antibodies (HMAb) to structurally conserved epitopes of HCV. Such antibodies can be found in a high proportion of patients and are useful, for example, in the diagnosis and therapy of HCV infection, including being useful in the identification of patients expected to benefit from certain therapeutic strategies.

2. Background

Hepatitis C virus (HCV) is an enveloped virus the genetic information for which is encoded in a 9.5 kb positive strand RNA genome. A highly conserved noncoding region of 341 bp is localized at the 5'-end of this viral genome, which is followed by a long open-reading frame coding for a polyprotein of approximately 3,010 amino acids. Two putative envelope glycoproteins E1 (gp35) and E2 (gp72) have been identified with 5 or 6 and 11 N-linked glycosylation sites, respectively. A high level of genetic variability is associated with the envelope genes. This variability is highly accentuated at the 5'-end of the E2 gene, where two hypervariable regions termed HVR1 and HVR2, have been described. Antibodies to HVR1 appear to mediate virus neutralization in cell culture and chimpanzee protection studies (Farci et al., 1996 Proc. Natl. Acad. Sci. USA 93:15394 15399; Shimizu et al., 1994J. Virol. 68:1494 1500; each of which is incorporated herein by reference). Unfortunately, antibodies to HVR1 tend to be isolate specific and over time drive the replication of new viral variants that the existing immune response does not recognize (Farci et al., 1994 Proc. Natl. Acad. Sci. USA 91:7792 7796; Weiner et al., 1992 Proc. Natl. Acad. Sci. USA 89:3468 3472; Kato et al., 1993 J. Virol. 67:3923 3930; each of which is incorporated herein by reference), although progress has been made at inducing a broader immune response to HVR1 related sequences (Puntoriero et al., 1998 EMBO Journal 17:3521 3533; incorporated herein by reference). HCV envelope antigens appear to be highly immunogenic when expressed in glycosylated forms (da Silva Cardoso et al., 1997 Ann. Hematol. 74:135 7; incorporated herein by reference). Preliminary data suggest the existence of conserved epitopes within the E2 protein (Lesniewski et al., 1995 J. Med. Virol. 45:415 22; incorporated herein by reference). The existence of neutralizing antibodies in serum from infected patients has been proposed.

Studies using HCV E1 E2 proteins expressed in mammalian cells have shown that infected individuals have an antibody response to HCV E2 composed in part to epitopes that are conformational in nature (Harada et al., 1994 J. Gen. Virol. 76:1223 1231; incorporated herein by reference). Studies involving the isolation of human monoclonal or recombinant antibodies to HCV E2 protein showed that a substantial fraction of these antibodies recognize conformational epitopes (da Silva Cordoso et al., 1998 J. Med. Virol. 55:28 34; Burioni et al., 1998 Hepatology 28:810 814; Habersetzer et al., 1998 Virology 249:32 41; each of which is incorporated herein by reference). As to biological function of these domains, investigators have employed surrogate assays to provide insights into virus neutralization since the virus cannot be grown, in vitro (Houghton, Hepatitis C viruses. In Fields, Knipe, Howley (eds) Virology. Lippincott-Raven, Philadelphia, pp. 1035 1058; incorporated herein by reference). One surrogate assay, the neutralization of binding (NOB) assay, evaluates the ability of a given antibody or serum to prevent the association of HCV E2 protein with a human T-cell line (Rosa et al., 1996 Proc. Natl. Acad. Sci. USA 93:1759 1763; incorporated herein by reference). The finding that serum antibodies obtained from chimpanzees protected by vaccination were strongly positive in the NOB assay provides support for the relevance of the assay as a measure of virus neutralization activity (Rosa et al., supra; Ishii et al., 1998 Hepatology 28:1117 1120; each of which is incorporated herein by reference).

The human tetraspannin cell surface protein CD81 (TAPA-1, for review see Levy et al., 1998 Ann. Rev. Immunol. 16:89 109; incorporated herein by reference) is the target protein bound by HCV E2 in the NOB assay (Pileri et al., 1998 Science. 282:938 941; incorporated herein by reference). Furthermore, human CD81 binds to free virions, and subsequently is a possible receptor for HCV (Pileri et al., supra). Using HCV 1a E2 proteins, several human monoclonal antibodies to HCV E2 protein have been reported to inhibit the interaction of HCV E2 with human cells (Burioni et al., 1998 Hepatology 28:810 814; Habersetzer et al., 1998 Virology 249:32 41; each of which is incorporated herein by reference). However, little is known about the conservation of the epitopes recognized by the NOB positive antibodies in HCV E2 proteins of different genotypes.

Other approaches to detection of and protection against HCV include the development of peptide mimetics. As an example, peptide mimetics of Hepatitis type A and C viral proteins have been created through production of randomly generated synthetic and phage-display peptide libraries for use in detection assays and vaccination therapies (Mattioli et al., 1995 J. Virology 69:5294 5299; Prezzi et al., 1996 J. Immunol. 156:4504 4513; each of which is incorporated herein by reference). However, effective antibody binding of these mimotopes has only been compared to linearly defined viral epitopes. The sequential recombinant fusing of several linearly defined immunodominant HCV epitopes has been described for use in diagnostic assays (Chein et al., 1999 J. Clin. Microbiol. 37:1393 1397; incorporated herein by reference). However, this multiple-epitope fusion antigen designed from linear epitopes was not created to function in the same capacity as a conformational mimetic: it was not designed to interfere with binding to a target receptor.

It is therefore of substantial interest to identify neutralizing antibodies in serum from infected patients which may be used in diagnosis and passive immunotherapy, where the antibodies would originate from a human cell, and provide for neutralization of a broad spectrum of genotypes, particularly in a particular geographical area. Both breadth of reactivity to multiple HCV genotypes and the ability to interfere with the binding of HCV virions to susceptible cells would be key attributes for a therapeutically useful neutralizing antibody. Also of interest is the design of peptide and non-peptide (organic) structural mimetics of HCV envelope proteins.

Relevant Literature

References providing background information concerning HCV include Abrignani 1997 Springer Semin. Immunopathology 19:47 55; Simmonds, 1995 Hepatology 21:570:583; and Mahaney et al., 1994 Hepatology 20:1405 1411; each of which is incorporated herein by reference.

Da Silva Cardosa et al., 1998 J. Med. Virology 55:28 34 describe human monoclonal antibodies to HCV E1/E2. Habersetzer et al., 1998 Virology 249:32 41 describe human monoclonal antibodies capable of recognizing HCV E2 genotypes 1a and 1b. Burioni et al., 1998 report human recombinant Fabs for the HCV E2 protein. Deleersnyder et al., 1997 J. of Virology 71:697 704 describe an E2 reactive monoclonal antibody. Other references related to the use of antibodies to HCV include Burioni et al., 1998 Hepatology 28:810 814; Akatsuka, et al., 1993 Hepatology 18:503 510; DeLalla, et al., 1993 J. Hepatol. 18:163 167; Mondelli, et al., 1994 J. Virol. 68:4829 4836; Siemoneit, et al., 1994 Hybridoma 13:9 13; and Moradpour, et al., 1996 J. Med. Virol. 48:234 241; for producing human antibodies, Foung, et al., 1990 J. Immunol. Methods 70:83 90; Zimmerman, et al., 1990 J. Immunol. Methods 134:43 50; for producing modified antibodies using combinatorial libraries, Burton and Barbas, Dixon, F J (Ed.) Advances in Immunology, Vol. 57, Vi+391 p. Academic Press, Inc., San Diego, Calif., 191 280, 1994; Plaisant, et al., 1997 Res. Virol. 148 169; and Barbas and Burton, Monoclonal Antibodies from Combinatorial Libraries. Cold Spring Harbor Laboratory Course Manual, Cold Spring Harbor, N.Y., 1994. Each of the references cited in this paragraph is incorporated herein by reference.

An assay for antibodies binding to HCV E2 is described by Rosa et al., 1996 Proc. Natl. Acad. Sci. USA 93:1759 1763; incorporated herein by reference.

Vaccinia virus or baculovirus constructs having a portion of the HCV genome are described by Ralston et al., 1993 J. Virology 67:6733 6761 and Lanford et al., 1993 Virology 197:225 235; each of which is incorporated herein by reference.

SUMMARY OF THE INVENTION

One aspect of the present invention provides monoclonal antibodies, including human monoclonal antibodies, which bind to the dominant HCV types in major geographical areas. The dissociation constants for the antibodies to their epitopes are, for example, less than 10.sup.-7M, less than 10.sup.-8M, to less than 10.sup.-9M, and less than 10.sup.-10M. Specifically, a family of monoclonal antibodies binding to conformationally conserved epitopes of the HCV E2 protein is provided. Among the family are antibodies that bind to the dominant genotypes found in the United States, so as to be substantially pan-monoclonal antibodies in being able to bind to almost all cases of HCV infection, which have been diagnosed in the United States, as well as at least a substantial proportion of the cases in other geographic locales. The monoclonal antibodies find use in a variety of diagnostic assays. In addition, conformationally conserved expression of recombinant type 1 and type 2 HCV E2 proteins and fragments thereof are provided for use in assays, screening drugs, vaccines, diagnostic assays, and for other purposes. The inventive antibodies find use in passive immunotherapy strategies for reducing viral load of infected individuals and interfering with the infection of target cells. Antibodies recognizing conformationally dependent epitopes can also be used to provide a template for the rational design of peptide and conformationally-defined epitope mimetics (e.g., organic compounds, organometallic compounds, inorganic compounds, small molecules).

In a particularly preferred embodiment, the inventive antibodies are directed to conformational epitopes of the E2 or E1 protein of HCV. Conformational epitopes of E2 have been identified using a panel of monoclonal antibodies and a series of deletion constructs of E2. One group of antibodies has been found to bind to conformational epitopes between E2 amino acids 411 644 from HCV 1b. Antibodies of this group have been found to inhibit the interaction of E2 with CD81. Another group of antibodies has been found to bind to conformational epitopes between HCV 1b E2 amino acids 470 644. A third group of antibodies binds to conformational epitopes between HCV 1b E2 amino acids 470 644 but fails to inhibit the binding of E2 to CD81. A fourth group binds to conformational epitopes between HCV 1b E2 amino acids 644 661. In a particularly preferred embodiment, the conformational epitopes to which the antibodies are directed are conserved among HCV strains. The antibodies of the present invention may be combined with pharmaceutically acceptable excipients to provide pharmaceutical formulations.

Another aspect of the invention provides definition of conformational epitopes in HCV proteins, and further provided compositions and compounds containing such epitopes. For example, the present invention provides proteins, peptides, and small molecules comprising the conformational epitopes of HCV E2 protein. The peptides may be deletion constructs such as those in FIG. 23. The peptides may contain one or more conformational epitopes recognized by the antibodies of the present invention. In certain preferred embodiments, the proteins are strings of concatenated peptides at least one of which contains a conformational epitope of HCV. The peptides of the string may contain different conformational or linear epitopes of HCV or the peptides may contain the same epitope. The peptides of the string should preferably fold properly in order to display the conformational epitope substantially as it appears in nature. Such proteins and peptides may be used in formulating vaccines or used in diagnostic tests.

The present invention also provides a method for stratifying patients based on their immunological response to HCV and of identifying those patients likely to respond well to HCV immunotherapy. For example, a patient's serum may be used to test for the presence of antibodies directed against a particular epitope of HCV. If the patient does not have adequate levels of antibodies directed to such an epitope, human monoclonal antibodies directed against the epitope may be administered to the patient.

DEFINITIONS

"Animal": The term animal, as used herein, refers to humans as well as non-human animals, including, for example, mammals, birds, reptiles, amphibians, and fish. Preferably, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a primate, or a pig). An animal may be a transgenic animal.

"Antibody": The term antibody refers to an immunoglobulin, whether natural or wholly or partially synthetically produced. All derivatives and fragments thereof which maintain specific binding ability are also included in the term. The term also covers any protein having a binding domain which is homologous or largely homologous to an immunoglobulin binding domain. These proteins may be derived from natural sources, or partly or wholly synthetically produced. An antibody may be monoclonal or polyclonal. The antibody may be a member of any immunoglobulin class, including any of the human classes: IgG, IgM, IgA, IgD, and IgE. Derivatives of the IgG class, however, are preferred in the present invention.

"Peptide": According to the present invention, a "peptide" comprises a string of at least three amino acids linked together by peptide bonds. Peptide may refer to an individual peptide or a collection of peptides. Inventive peptides preferably contain only natural amino acids, although non-natural amino acids (i.e., compounds that do not occur in nature but that can be incorporated into a polypeptide chain; see, for example, http://www.cco.caltech.edu/.about.dadgrp/Unnatstruct.gif, which displays structures of non-natural amino acids that have been successfully incorporated into functional ion channels) and/or amino acid analogs as are known in the art may alternatively be employed. Also, one or more of the amino acids in an inventive peptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. In a preferred embodiment, the modifications of the peptide lead to a more stable peptide (e.g., greater half-life in vivo). These modifications may include cyclization of the peptide, the incorporation of D-amino acids, etc. None of the modifications should substantially interfere with the desired biological activity of the peptide.

"Polynucleotide" or "oligonucleotide": Polynucleotide or oligonucleotide refers to a polymer of nucleotides. The polymer may include natural nucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine), chemically modified bases, biologically modified bases (e.g., methylated bases), intercalated bases, modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose), or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages).

"Small molecule": As used herein, the term "small molecule" refers to organic compounds, whether naturally-occurring or artificially created (e.g., via chemical synthesis) that have relatively low molecular weight and that are not proteins, polypeptides, or nucleic acids. Typically, small molecules have a molecular weight of less than about 1500 g/mol. Also, small molecules typically have multiple carbon--carbon bonds. Known naturally-occurring small molecules include, but are not limited to, penicillin, erythromycin, taxol, cyclosporin, and rapamycin. Known synthetic small molecules include, but are not limited to, ampicillin, methicillin, sulfamethoxazole, and sulfonamides.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a Western blot indicating the expression of HCV E2 proteins by some of the vaccinia virus constructs described in this application. Cytoplasmic extracts were prepared from CV1 cells infected with wild type vaccinia virus and then transfected with pVOTE (wt) or recombinant pVOTE expressing HCV E2 of genotype 1a (Q1a) or 2b (Q2b). Cells were cultured for 24 hours in the presence (+) or absence (-) of the inducer IPTG. Extract corresponding to 2.times.10.sup.5 cells was fractionated by SDS PAGE and blotted onto nitrocellulose. HCV E2 protein was revealed by incubation with 1/500 diluted ascites fluid of mMab E2G using the ECL detection system (Amersham). Migration of molecular weight standards is indicated at right.

FIG. 2 (SEQ ID NOS: 9 12) describes sequences amplified from the central region of the HCV E2 vaccinia virus clones. Shown are the sequences of the central fragment for HCV E2 vaccinia constructs Q1a, Q 1b, Q2a, and Q2b as compared to representative sequences of the appropriate HCV genotypes. Accession numbers for the representative sequences of each genotype are as follows HCV 1A=M62321, HCV 1B=D10750, HCV2A=D00944, HCV 2B=D10988. Phylogenetic analysis was performed with CLUSTALV and DNAPARS program of the PHYLIP package.

FIG. 3 (SEQ ID NOS: 1 8) is a comparison of sequences of HCV: 1a, Q1a-FR, -1b, 1Q1b-FR, 2a, Q2a-FR, -2b, -Q2b-FR, using the most parsimonious tree found.

FIG. 4 shows a graph of the reactivity of HCV sera with HCV E2 proteins of different genotypes. HCV E2 protein expressed by 6.times.10.sup.5 HeLa cells infected with vaccinia virus Q1a .box-solid., Q1b .tangle-solidup., Q2a , Q2b .diamond-solid., or non recombinant vaccinia virus VWA .smallcircle. was captured onto wells coated with 500 ng of GNA lectin. Wells were washed and blocked, and bound protein was incubated with increasing dilutions (x axis) of genotyped HCV sera or an HCV negative serum (indicated above the graph). Values are the mean absorbance of replicate wells. Error bars indicate one standard deviation from the mean.

FIG. 5 is a bar graph indicating the reactivity of sera from individuals infected with HCV genotype 2b with HCV E2 proteins of multiple genotypes. Twelve sera from individuals infected with HCV genotype 2b (x axis) were titrated against HCV E2 proteins of genotypes 1a (dark blue bars), 1b (magenta bars), 2a (yellow bars), and 2b (light blue bars). The dilution of the serum that resulted in a mean specific absorbance (mean absorbance obtained with HCV E2 containing extract subtracted from the mean specific absorbence obtained with the VWA extract) of 0.5 is indicated on the y axis. This value was calculated from titration curve data analogous to that presented in FIG. 4.

FIG. 6 depicts a schematic of a direct binding assay to assess for antibodies that recognize conformational epitopes of HCV E2 envelope proteins employed in the experiments described in FIGS. 7, 8, and 9. GNA lectin is coated onto a solid surface and then added E2-containing protein extracts are captured by the lectin. Test antibodies are allowed to bind to the captured E2, the excess unbound is removed, and bound antibody is detected with a labeled secondary antibody.

FIG. 7 is a bar graph of the reactivity of HCV HMAbs to HCV E2 captured by lectins. Proteins from cytoplasmic extracts of 6.times.10.sup.5 cells infected with wild type (Bars labeled VWA) or HCV 1a E2 (HCV 1a) (bars labeled HCV) expressing vaccinia virus were applied to microtiter plates coated with 500 ng of Galanthus nivalis (GNA) or Tiriticum vulgaris (WGA). Captured proteins were incubated with 5 .mu.g/ml of the indicated HMAbs (x axis). R04 is an isotype-matched control. Bound HMAb was detected with anti-human antibody-alkaline phosphatase and appropriate substrate. Bars indicate the mean OD value of replicate wells. Error bars indicate one standard deviation from the mean.

FIG. 8 shows graphs of HCV antibody reactivity with E2 protein of divergent HCV genotypes. HCV E2 proteins expressed by 6.times.10.sup.5 HeLa cells infected with vaccinia virus Q1a .box-solid., Q1b .tangle-solidup., Q2a , Q2b .diamond-solid. was captured onto wells coated with 500 ng of GNA lectin. Wells were washed and blocked, and bound protein was incubated with the indicated HCV HMAbs (HMAb identified above each of FIGS. 9A 9J) and control HMAb (R04) FIG. 9K to a CMV protein (Ward, et al., 1995, Proc Natl Acad Sci USA. 92:6773 6777; incorporated herein by reference). Values are the mean specific binding (extracts of cells infected with vaccinia virus expressing HCV E2 protein--wt vaccinia extracts) of replicate wells. Reactivity of HCV and control HMAbs with proteins from wt vaccinia virus infected cells did not exceed an absorbance of 0.04. Error bars indicate one standard deviation from the mean.

FIG. 9 is a bar graph showing the reactivity of HCV HMAbs with native (NAT) and denatured (DNT) HCV 1b E2 protein. Cytoplasmic extract derived from 6.times.10.sup.5 HeLa cells infected with vaccinia virus Q1b and VWA or VWA alone were either left untreated (blue bars) or denatured by incubation with 0.5% SDS and 5 mM dithiothreitol for 15 minutes at 56.degree. C. (yellow bars). After treatment, proteins were diluted 1:5 in BLOTTO and captured onto wells coated with 500 ng of GNA lectin. Wells were washed and blocked, and bound protein was incubated with the indicated concentration of HCV HMAbs and control HMAb (R04). Bound antibody was detected with anti-human IgG alkaline phosphatase conjugate and PNPP. Color development was allowed to proceed for 45 minutes. Values for native and denatured HCV 1b are the mean signal obtained from replicate wells. Signals from single wells of native and denatured proteins derived from VWA infected HeLa cells were indistinguishable and also averaged (red bars). Error bars indicate one standard deviation from the mean.

FIG. 10 depicts a schematic of the competition binding analysis employed in the experiments described in FIGS. 11, 12, and 13. GNA lectin is coated onto a solid surface and then added E2-containing protein extracts are captured by the lectin. Competing antibodies are allowed to bind to the captured E2 before removing unbound excess and adding labeled test antibody.

FIG. 11 is a bar graph of a competition analysis using HCV HMAb CBH-5. HCV E2 protein from cytoplasmic extracts of HeLa cells infected with vaccinia virus Q1a (blue bars) or Q1b (red bars) was captured with 500 ng of GNA. Bound HCV E2 was detected with 5 .mu.g/ml of biotinylated CBH-5 in the presence of 25 .mu.g/ml of the indicated HMAbs (x axis). Results are compared to binding of biotinylated CBH-5 in the absence of any competitor. Bars indicate the mean value obtained from replicate wells. Error bars indicate one standard deviation from the mean.

FIG. 12 is a competition analysis showing the ability of the HCV HMAbs to interfere with the binding of HMAb CBH-2 to HCV E2 proteins of multiple genotypes. HCV E2 protein from cytoplasmic extracts of HeLa cells infected with vaccinia virus Q1a (Blue bars), Q1b (red bars), Q2a (yellow bars), or Q2b (light blue bars) was captured with 500 ng of GNA lectin. The HMAbs CBH-4D, -4B, and -17 were only evaluated with HCV 1a or 1b E2 protein due to their limited reactivity to genotype 2 E2 proteins. Bound HCV E2 was detected with 2 .mu.g/ml of biotinylated CBH-2 in the presence of 20 .mu.g/ml of the indicated HMAbs (x axis). The bars indicate the binding observed in the presence of the indicated antibody relative to binding of biotinylated CBH-2 to HCV E2 in the absence of any competing antibody (y axis). R04 is a control HMAb that recognizes a cytomegalovirus protein. Bars indicate the mean value obtained from replicate wells. Error bars indicate one standard deviation from the mean.

FIG. 13 is a competition analysis showing that HCV HMAb CBH-7 recognizes a unique epitope. HCV E2 protein from cytoplasmic extracts of HeLa cells infected with vaccinia virus Q1a (blue bars) or Q1b (red bars) was captured with 500 ng of GNA lectin. Bound HCV E2 was detected with 2 .mu.g/ml of biotinylated CBH-7 in the presence of 20 .mu.g/ml of the indicated HMAbs (x axis). The bars indicate the binding observed in the presence of the indicated antibody relative to binding of biotinylated CBH-7 to HCV E2 in the absence of any competing antibody (y axis). R04 is a control HMAb that recognizes a cytomegalovirus protein. Bars indicate the mean value obtained from replicate wells. Error bars indicate one standard deviation from the mean.

FIG. 14 depicts a schematic for assessing the ability of antibodies to block CD81 binding to E2 proteins as employed in the experiments described in FIG. 1. Recombinant CD81 is coated onto a solid surface. E2-containing protein extracts are then either added directly, or after preincubation with the test antibody. Bound test antibody-E2 complexes are detected using an appropriate labeled secondary antibody.

FIG. 15 is a bar graph that demonstrates that a subset of HCV HMAbs react with HCV E2 when bound to CD81-LEL. Extracts from BSC-1 cells infected with recombinant vaccinia virus expressing HCV E2 proteins were combined with 5 .mu.g/ml of the indicated HMAbs (x axis) in a total volume of 100 .mu.l and incubated in microtiter plate wells coated with 100 ng of a GST CD81-LEL fusion protein or non-recombinant GST overnight. Wells were washed and bound antibody was detected using an appropriate alkaline-phosphate conjugated secondary antibody and PNPP substrate as further described in Example 6. Values are the mean OD value of antibody captured by CD81 divided by the mean OD value for antibody captured by GST in the presence of 1a (purple bars), 1b (red bars), 2a (yellow bars), or 2b (green bars) E2 protein. OD values obtained from wells coated with GST ranged between 0.021 and 0.081.

FIG. 16 depicts a schematic for assessing the ability of antibodies to block CD81 binding to HCV virions as employed in the experiments described in FIG. 17. Recombinant CD81 is coated onto a solid surface. HCV virions are preincubated with test antibodies, and then allowed to bind to immobilized CD81. Detection of bound HCV virions is measured by quantitative PCR.

FIG. 17 shows a bar graph demonstrating that HMAbs CBH-2 and CBH-5 inhibit binding of HCV virions to CD81. The number of HCV RNA molecules bound to polystyrene beads (x axis) after HCV 1a chimpanzee serum was combined with 10 .mu.g of the indicated antibodies (y axis) and then allowed to bind to beads coated with CD81-LEL as described in Example 7.

FIG. 18 is a bar graph that shows that HMAb CBH-4G can be employed to detect the presence of antibodies that inhibit binding of HCV E2 to CD81. HCV 1a E2 protein derived from extracts of BSC-1 cells infected with vaccinia virus Q1a was incubated with 4 .mu.g/ml of a biotinylated preparation of HMAb CBH-4G for 20 minutes at 4.degree. C. A 50 .mu.l aliquot of the E2-CBH-4G complexes were then added to wells coated with either 500 ng of GNA (blue bars) or 100 ng of GST-CD81-LEL (red bars) to which 50 .mu.l of a 40 .mu.g/ml of the indicated antibodies (x axis) was added. R04 is a control HMAb that recognizes a cytomegalovirus protein. After an overnight incubation at 4.degree. C. the wells were washed and bound biotinylated CBH-4G detected as described in Example 8. The bars indicate the mean signal obtained from duplicate wells in the presence of the indicated antibody relative to the signal obtained in the absence of any competing antibody. Error bars indicate one standard deviation from the mean.

FIG. 19 is a bar graph that shows that HMAb CBH-4G can be employed to detect the presence of antibodies that inhibit binding of HCV E2 to CD81 in sera from HCV infected individuals. HCV 1a or 2b E2 protein derived from extracts of BSC-1 cells infected with vaccinia virus Q1a or Q2b was incubated with 4 .mu.g/ml of a biotinylated preparation of HMAb CBH-4G for 20 minutes at 4.degree. C. The four sera at left were tested with HCV 1a E2 protein, the four sera at right were tested with HCV 2b E2 protein. The E2-CBH-4G complexes were then added to wells coated with either 500 ng of GNA (blue bars) or 100 ng of GST-CD81-LEL (red bars) in the presence of a 1/500 dilution of the indicated sera from genotyped HCV infected (1a or 2b) or uninfected (NEG) individuals (x axis). After an overnight incubation at 4.degree. C., the wells were washed, and bound biotinylated CBH-4G was detected as described in Example 8. The bars indicate the mean signal obtained from duplicate wells in the presence of the indicated serum (final dilution 1/1000) relative to the signal obtained in the absence of any competing serum. Error bars indicate one standard deviation from the mean.

FIG. 20 is a cartoon of the competition assay. Plates are first coated with GNA lectin which is used to capture full-length intracellular E2 onto microtiter plates by binding of CHO moieties to GNA lectin. Competing HMAb are contacted with the GNA-captured E2. Biotinylated test HMAb is added to the plates, and binding of the biotinylated test HMAb to E2 is detected using a streptavidin-AP conjugate. Inhibition of binding of test HMAb suggests epitopes within same antibody binding domain.

FIG. 21 shows competition analysis of four HCV human monoclonal antibodies. HCV Q1b E2 protein was captured onto GNA lectin coated microtiter plates. Biotinylated test antibody (indicated above each panel) at 2 .mu.g/ml was added to wells containing the indicated concentration .alpha.-axis) of competing human monoclonal antibody. Bound biotinylated test antibody was detected using streptavidin alkaline phosphatase conjugate. Signal obtained in the presence of competing antibody was expressed as the percent of signal obtained by the biotinylated test antibody relative to the signal obtained in the absence of competing antibody (y-axis). The points indicate the mean value obtained from two replicate wells. The bars indicate one standard deviation from the mean. Competing antibodies are identified in the key at left.

FIG. 22 shows the results of a human monoclonal competition analysis. Results are the mean percent binding of test antibody relative to wells without any competing antibody. Results are the mean values obtained from 2 5 separate experiments. Both genotype 1a and 1b E2 proteins were tested. ND=not done.

FIG. 23 depicts HCV E2 deletion constructs described herein. The names of the E2 constructs are provided at left. Sequences derived from the vector pDisplay are indicated as solid black bars. The positions of the HA epitope and the c-myc epitope present in the pDisplay vector are also indicated. Sequences derived from HCV 1b E2 are indicated as white boxes. Sequences derived from HCV 1b E2 are indicated as light gray boxes. Numbering of the X-axis (below) is according to the polyprotein of the HCV-1 isolate.

FIG. 24 shows Western blot analysis of HCV E2 deletion constructs indicating that the constructs are efficiently expressed. The indicated HCV E2 constructs (above lanes) were transfected into HEK-293 cells. Twenty-four hours after transfection cytoplasmic extracts were prepared and fractionated via SDS-PAGE. The fractionated proteins were transferred to nitrocellulose membranes and incubated with either rat monoclonal antibody to the HA epitope (HA rMAb) or a control HMAb to a CMV protein (control). Bound antibody was detected with the appropriate AP conjugated antisera. HEK=mock-transfected HEK-293 cells. The migration of molecular weight markers are indicated at left.

FIG. 25 shows reactivity of certain inventive human monoclonal antibodies with the various HCV E2 deletion constructs. HEK-293 cells were mock transfected (white bars) or transfected with the indicated HCV E2 constructs (see keys each graph). Twenty four hours post transfection cytoplasmic extracts were prepared and equivalent aliquots were captured onto GNA lectin coated microtiter plates as described above. The captured E2 proteins were then incubated with the indicated HCV HMAb (x-axis) and the amount of bound antibody was determined. Bars represent the mean-absorbance value obtained from duplicate wells. Error bars indicate one standard deviation from the mean.

FIG. 26 shows graphs demonstrating that sera from HCV infected individuals have variable levels of antibodies that inhibit CBH-2 and CBH-7. Homologous HCV E2 proteins were captured onto wells and incubated with the increasing dilutions of HCV 1a, 1b, 2a, or 2b sera. Values are the specific inhibition of binding of biotinylated CBH-2 or CBH-7 obtained with individual sera. The mean percent inhibition (y-axis) obtained from duplicate determinations at a given dilution (x-axis) are plotted. The mean specific inhibition obtained for eight negative sera are also presented (genotypes of E2 proteins employed are indicated). Error bars on negative sera indicate one standard deviation from the mean.

FIG. 27 shows scatterograms demonstrating that sera from HCV infected individuals have variable levels of antibodies that inhibit CBH-2 and CBH-7. Scattergram showing percentage of test HMAb inhibition. HCV sera of the indicated genotype (x-axis) or control sera (NEG) were diluted 1:200 and incubated with biotinylated test HMAb (indicated above graph) in wells coated with genotyped matched E2 proteins. Binding of test HMAb was detected using streptavidin-conjugated-AP. Results obtained were compared to binding of test HMAb in absence of competitor. Each symbol indicates results obtained with an individual serum. The line indicates the median percent inhibition. The dotted line indicates the cutoff for calling a serum positive for the presence of the test HMAb.

FIG. 28 is a histogram of CBH-2 inhibitory titers obtained from a panel of 74 individuals with chronic hepatitis. The CBH-2 inhibitory titers obtained with individual serum were segregated into 20 bins of 100 and 1 bind for all titers >2000. The bars indicate the number of sera having a CBH-2 inhibitory titer within a given bin. Numbers of HCV 1a/1b sera are indicated in black. Number of HCV 2a/2b sera are indicated in gray. The number of sera with low (<200), intermediate (200 1000), and high (>1000) inhibitory titers are indicated below the graph.

FIG. 29 is a histogram of CBH-7 inhibitory titers obtained from a panel of 74 individuals with chronic hepatitis. The CBH-7 inhibitory titers obtained with individual serum were segregated into 20 bins of 100 and 1 bin for all titers >2000. The bars indicate the number of sera having a CBH-7 inhibitory titer within a given bin. Numbers of HCV 1a/1b sera are indicated in black. Number of HCV 2a/2b sera are indicated in gray. The number of sera with low (<200), intermediate (200 1000), and high (>1000) inhibitory titers are indicated below the graph.

DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS OF THE INVENTION

Monoclonal antibodies, particularly human monoclonal antibodies ("HMAbs"), are provided which bind to one or more hepatitis C virus genotypes, which antibodies find use for diagnosis and therapy. A panel of human monoclonal antibodies (HMAbs) from peripheral B-cells of an individual with asymptomatic HCV infection and having a high serum neutralization of binding titer were produced and characterized. Eleven HMAbs to HCV E2 have been produced. One group of antibodies binds to the genotypes of HCV types 1 and 2, while other antibodies bind to fewer than this group of genotypes. HCV types 1 and 2 together are the dominant virus types encountered in the western hemisphere and other geographic locations. The antibodies bind to conformational epitopes which are conserved across virus types and genotypes. The antibodies bind to HCV E2 proteins of genotypes 1a, 1b, 2a, and 2b and a subset of these antibodies inhibit the interaction of these E2 proteins with human CD81. By virtue of the variety of binding profiles of the antibodies, diagnostic assays may be employed which will detect a plurality of types and genotypes, so as to provide a pan-anti-HCV antibody for HCV encountered in the United States, while at the same time being able to dissect individual genotypes by subtractive analysis. In addition, the antibodies being human may be used for passive immunization, as protective therapy for individuals at risk for HCV or as a therapy for people who are seropositive for HCV.

The HMAbs of the invention offer several advantages over existing HMAbs against HCV. Because non-homologous primary amino acid sequences may still define immunologically identical tridimensional protein structures, HMAbs binding to structurally conserved epitopes can recognize multiple, sequentially divergent HCV genotypes in native conformation, whereas antibodies recognizing only linear or denatured epitopes may not. In particular, conformationally dependent anti-HCV E2 HMAbs may effectively interfere with the interaction of native HCV virus and its cellular target receptors. Using conformationally dependent HMAbs to actively interfere with the ability of native HCV virus to bind to target cell receptors such as CD81 has specific therapeutic application for reducing viral load in infected individuals, and preventing infection or re-infection of organs in non-infected individuals, particularly in recent organ transplant recipients. Certain subsets of the HMAbs interfere with E2-associated viral infection by mechanisms other than preventing direct interaction with CD81. This subset of antibodies interferes with viral infectivity by a number of possible mechanisms, including preventing E2 binding to co-receptor proteins, conformational changes in E1 and/or E2 proteins necessary for target cell binding, E1, and E2-mediated viral fusion to target cells, and uncoating of HCV virions. Because they bind distinct conformational epitopes, the subset of HMAbs that directly interferes with E2 binding to CD81 complements HMAbs in the subset that interfere with infectivity by other mechanisms for both therapeutic and diagnostic applications.

HMAbs which recognize conformationally-defined viral epitopes and interfere with virus/target receptor interaction, and viral conformational epitopes which bind to such HMAbs, may also serve as templates for rationally designing peptide and other structural mimics of the viral epitopes. Structural molecular mimics defined by these conformationally dependent anti-HCV HMAbs find use in their ability to block binding of the native virus to target receptors by binding to the target receptor themselves.

By producing human monoclonal antibodies, it is possible to directly analyze the human immune response to HCV. Importantly, by using human monoclonal antibodies, immune responses against the antibodies themselves as foreign antigens are minimal, whereas vigorous immune responses are generated against monoclonal antibodies produced from non-human sources, because they are recognized as foreign antigens. Selecting for HMAbs that recognize conserved viral conformational epitopes affords broader and more effective therapeutic application of these reagents for ameliorating or preventing HCV infection than antibodies able to bind only linear or denatured epitopes. All previous antibodies described as having the property of preventing HCV infection or uptake into target cells recognize a highly variable sequence of HCV E2 known as the hypervariable region. In contrast, the antibodies described above recognize conformational epitopes, the majority of which are highly conserved HCV E2 proteins of multiple different genotypes. Thus the antibodies described herein have the advantage that they are active against a much wider range of HCV isolates than previously described neutralizing antibodies. An additional advantage is that the high conservation of the epitopes recognized by the antibodies described herein indicates that these antibodies recognize sequences with functional and/or structural significance within the HCV E2 protein. Thus peptides or small molecules isolated with these antibodies have a high probability of being targeted to functional regions within HCV E2. This is not true for other HVC antibodies described to date.

Of the detection antibodies described, CBH-4G has essentially equal reactivity to HCV E2-CD81 complexes of multiple HCV genotypes, whereas CBH-4B recognizes HCV genotypes 1a and 1b. The level of interfering antibodies present in HCV antisera has also been shown to be quite low. Therefore they provide a straight forward means of assaying the level of neutralizing antibodies present in a sample in a microtiter plate format without resorting to multiple flow cytometric analyses.

Mouse/human heteromyeloma cell lines expressing monoclonal antibody CBH-4B