Title:  Methods for the preparation of hepatitis C virus multiple copy epitope fusion antigens

United States Patent:  6,514,731

Issued:  February 4, 2003

Inventors:  Valenzuela; Pablo D. T. (Berkeley, CA); Chien; David Ying (Alamo, CA)

Assignee:  Chiron Corporation (Emeryville, CA)

Appl. No.:  653226

Filed:  May 24, 1996

Human hepatitis C virus (HCV) has been identified as the aetiological agent of non-A, non-B hepatitis (NANBH). HCV viruses display considerable genotypic and phenotypic heterogeneity. Thus, there is considerable need in the art for more sensitive reagents that facilitate the detection of HCV variants. The genome of hepatitis C virus (HCV) consists of seven functional regions: the core, E1, E2/NS1, NS2, NS3, NS4, and NS5 regions. An attempt was made to improve the sensitivity of anti-HCV assays by developing multiple copy epitope fusion antigens (MEFAs) which incorporate the major immunodominant epitopes from the functional regions of the HCV genome. These MEFAs are encompassed by the following generic structural formula: (A)_x --(B)_y --(C)_z. This formula represents a linear amino acid sequence comprising multiple copies of one HCV epitope (A) linked to multiple copies of another HCV epitope (B) which in turn is linked to multiple copies of yet another HCV epitope (C). Expression vectors carrying nucleic acid sequences comprising MEFA antigens carrying multiple copies of epitopes derived from the viral core, E1, E2, NS3, NS4, and NS5 regions were prepared. The resultant MEFA antigens were expressed, purified, and employed in suitable immunoassays for the detection of HCV-specific antisera. These antigens provide excellent sensitivity and specificity for the detection of HCV.

DETAILED DESCRIPTION OF EMBODIMENTS

Before the present multiple epitope fusion proteins, immunoassays and method for producing and using such are described, it is to be understood that this invention is not limited to the particular amino acid sequences, immunoassays or methods of production as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting since the scope of the present invention will be limited only by the appended claims.

Unless defined otherwise all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any method and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference for the purpose of disclosing and describing the particular technology which the publication is cited in connection with.

Definitions

The term "multiple copy" shall mean that a sequence of amino acids which contains at least five and not more than 1,000 amino acids in a linear fashion is repeated two or more times within a linear molecule. The repeating sequence need not be directly connected to itself, is not repeated in nature in the same manner, and further may be present within a larger sequence which includes other amino acids not repeated or "copied." The sequence of at least five and not more than 1,000 amino acids comprises an epitope as defined below. For the purposes of this invention, a "copy" of an amino acid sequence may be either an exact sequence copy or a sequence which corresponds to the same epitope of a different viral strain, i.e. copies are either exact copies or sequences which are "equivalent antigenic determinants" as defined below.

The term "epitope" shall mean a sequence of at least five, and not more than 1,000 acids connected in a linear fashion, which amino acids, by themselves or as part of a larger sequence, bind to an antibody generated in response to such sequence.

The term "conformational epitope" shall means a recombinant epitope having structurel features native to the amino acid sequence encoding the epitope within the full length natural protein. Native structural features include, but are not limited to, glycosylation and three dimensional structure. Generally, a conformational epitope is added to the MEFA-containing immunoassay mixture to enhance assay sensitivity and selectivity. Preferably, a recombinant conformational epitope is expressed in a cell from which it is extractable under conditions which preserve its desired structural features, e.g. without denaturation of the epitope. Such cells include bacteria, yeast, insect, and mammalian cells. Preferably, the cell in which a conformational epitope is expressed is a mammalian cell, such as a chinese hamster ovary cell (CHO). Expression and isolation of recombinant conformational epitopes from the E1 and E2 regions of HCV are described in WO 96/04301, WO 94/01778, WO 95/33053, WO 92/08734, which applications are herein incorporated by reference in their entirety.

The term "expression cassette" shall mean a DNA sequence which contains a coding region operably linked to suitable control sequences capable of effecting expression of the coding region in a compatible host. Expression systems invariably comprise a promoter, but, depending on the host intended, may contain additional critical DNA such as ribosome binding site or CAP site, termination sequence, and optional enhancer sequences upstream from the promoter or in other operable locations. The recombinant expression cassettes of the invention herein comprise a DNA of the invention encoding a MEFA operably linked to additional DNA sequences that are capable of effecting its expression. The expression cassette may reside on a transfer vector such as a plasmid or other vector that is self-replicating independently of the chromosome of the host cell, or may be constructed so that when inserted into a host cell it is able to integrate into the chromosome.

The term "equivalent antigenic determinant" shall mean an antigenic determinant from different sub-species or strain of a given organism e.g., a different strain of a virus such as strains 1, 2, and 3 of hepatitis C virus. More specifically for a virus such as hepatitis C, epitopes are known, such as 5-1-1, and such epitopes vary between the known strains 1, 2, and 3. Thus, the epitope 5-1-1 from the three different strains are equivalent antigenic determinants and thus are "copies" even though their sequences are not identical. In general the amino acid sequences of equivalent antigenic determinants will have a high degree of sequence homology, e.g., amino acid sequence homology of more than 30%, preferably more than 40%.

The term "tracer" shall mean any detectable marker molecule attachable to an epitope or a MEFA. Attachment is preferably by covalent means. Detectable marker molecules useful as tracers in the invention include, but are not limited to, dimethyl acridinium ester (DMAE), a chromophore, biotin, strepavidin, an antibody, an antigen, enzymes fluorogenic compounds, rhodamine compounds, fluorescein, FITC, and the like.

Producing Immunoassays--General

Highly sensitive and selective immunoassays can be produced using the multiple epitope fusion antigens of the present invention. In order to produce such immunoassays it is first necessary to identify a target for which a sample is to be assayed, e.g., assay for a particular virus in a body fluid sample. After identifying the virus of interest, the preferred immunodominant epitopes of the virus are isolated, sequenced and nucleotide sequences encoding the amino acid sequences of the epitopes are determined and produced. The nucleotide sequences encoding the amino acid sequences can be fused together using standard recombinant methodology.

The fused sequence must include at least two copies of nucleotide sequences that encode a given epitope. The nucleotide sequence is then placed within an expression cassette and a suitable host is transformed with the cassette. The host is allowed to express the sequences to provide the multiple copy epitopes (multiple epitope fusion antigen, MEFA). The multiple copy epitopes produced are then purified, for example, by affinity chromatography, which process is expedited to a certain degree due to the presence of the multiple copies of a given epitope. The purified MEFAs are then coated onto the surface of the substrate for ELISA-type assays. Alternatively, the purified MEFAs are attached to a detectable marker tracer molecule for detection of antibody binding, such as in a chemiluminescence assay (CLIA).

The essence of the invention is the purified multiple copy epitopes, i.e., purified fusion proteins that include multiple copies of a given epitope fused, in a linear fashion in nature, to other epitopes that are not normally connected to each other in this fashion (MEFAs). The purified epitopes are encompassed by the general structural formula (I) as follows: (A)_x--(B)_y --(C)_z, which represents a linear amino acid sequence, B is an amino acid sequence of an epitope or cluster of epitopes and each B contains at least five and not more than 1,000 amino acids, y is an integer of 2 or more, A and C are each independently an amino acid sequence of an epitope or cluster of epitopes not immediately adjacent to B in nature, and x and z are each independently an integer of 0 or more wherein at least one of x and z is 1 or more. When each of x, y, or z is greater than 1 or when each of x, y, and z are greater than 1, the multiple copies of A, B and C may be identical, i.e., each copy of A (different from B and C) is the exact same amino acid sequence, each copy of B (different from A and C) is the exact same amino acid sequence, and each copy of C (different from A and B) is the exact same amino acid sequence. Alternatively, each A, B or C copy may be an equivalent antigenic determinant from different strains of the same virus. Thus, for example if y is 3, each B may be an identical amino acid sequence or three different sequences from equivalent antigenic determinants from HCV strain 1, 2, and 3. The invention may utilize genetic material encoding known epitopes or groups of epitopes by connecting the material in a nucleic acid construct that produces a multiple copy epitope of the formula (I).

HCV antibody capture assays in which the individual single epitopes are coated on a solid support are less sensitive than capture assays in which a chimeric multiple epitope polyprotein, such as (C25) containing epitopes from the immunodominant core, c33c (NS3), and c100 (NS4) region sequences (Chien, D. Y., et al (1992) Proc. Natl. Acad. Sci. USA 89:10011-10015, herein incorporated by reference), is coated on a solid support. In turn, a capture assay using the C25 chimeric polyprotein is less sensitive than an HCV antibody capture assay using a MEFA of the invention, which MEFA contains multiple copies of at least one epitope and at least one copy is from a different HCV strain. Thus, a preferred MEFA of the invention having the general formula Ax-By-Cz, contains more than one copy of an epitope (i.e., y is an integer of 2 or more), and at least one of the epitopes of B is a different equivalent antigenic determinant (e.g. an epitope from a different pathogen strain).

The invention disclosed herein utilizes recombinant DNA technology and protein engineering to design a recombinant polyprotein which fuses a variety of different immunodominant epitopes from a variety of pathogens or pathogen strains as the chimeric antigen for immunoassay development. Further, the invention utilizes multiple copies of selected epitopes from structural as well as non-structural coding regions of a gene combined and expressed as a recombinant polyprotein to significantly improve the sensitivity and selectivity of an immunoassay.

Epitopes used in making a multiple copy epitope of the invention can be from a variety of different organisms. For example, the epitope may be an amino acid sequence from bacteria, protozoa, virus, rickettsiae, parasite or fungus. A preferred embodiment of the invention uses epitopes that are extracted from a bacteria or virus, with particularly preferred epitopes being those obtained from a virus, such as from human immunodeficiency virus and, most preferably, from hepatitis C virus.

It is well known that any given organism varies from one individual organism to another and further that a given organism such as a virus can have a number of different strains. For example, hepatitis C virus includes at least strains 1, 2, and 3. Each of these strains will include equivalent antigenic determinants. More specifically, each strain will include a number of antigenic determinants that will be present on all strains of the virus but, will be slightly different from one viral strain to another. For example, hepatitis C includes the antigenic determinant known as 5-1-1 (in the NS3 region of the viral genome). This particular antigenic determinant appears in three different forms on the three different viral strains of hepatitis C. Accordingly, in a preferred embodiment of the invention all three forms of 5-1-1 appear on the multiple epitope fusion antigen of the invention. A MEFA of the invention has the above structural formula I, wherein y is 3 and thus each of the three "Bs" are equivalent antigenic determinants of 5-1-1 taken from the three different viral strains of hepatitis C.

The multiple copy epitope of the present invention can also include multiple copies which are exact copies of the same epitope. For example, it is desirable to include two copies of an epitope from the core region of hepatitis C. A particularly preferred embodiment of the present invention is the multiple copy epitope as shown within FIG. 3. This multiple copy epitope includes two exact copies of an epitope from the core region and three copies of an epitope from the 5-1-1 region, which copies are equivalent antigenic determinants meaning that they are antigenic determinants taken from the three different viral strains of hepatitis C. In general, equivalent antigenic determinants have a high degree of homology in terms of amino acid sequence which degree of homology is generally 30% or more or more preferably 40% or more.

Producing HCV Immunoassays

Highly selective and sensitive immunoassays generally contain major immunodominant epitopes of the pathogen suspected of infecting a patient. Previously, immunoassays made use of individual epitopes to bind anti-HCV antibodies in biological samples.

For the virus HCV, major immunodominant linear epitopes were identified from the core, NS3 (nonstructural), NS4, and NS5 regions of the virus polyprotein. Sallberg et al. assayed HCV core protein and putative matrix proteins against human serum samples containing antibodies to HCV and defined several immunodominant regions within the HCV proteins (Sallberg, M. et al. (1992) J. Clin. Microbiol. 30:1989-1994). Protein domains of HCV-1 polyproteins including domains C, E1, E2/NS1, NS2, NS3, NS4, and NS5 were identified and their approximate boundaries provided by Chien and Rutter (Chien, D. Y. and Rutter, W., WO 93/00365, international publication date Jan. 7, 1993, herein incorporated by reference in its entirety). Kotwal et al. designed individual polypeptides having sequences derived from the structural region of HCV in order to obtain an immunodominant epitope useful in testing sera of HCV patients (Kotwal, G. J., et al. (1992) Proc. Natl. Acad. Sci. 89:4486-4489).

Serologically definable subtypes of HCV were identified by Chien et al. as viral subtypes exhibiting varied antigenicity (presented at the Third International Hepatitis Meeting, Tokyo, May, 1993 and in Chien, D. Y. et al. (1994) Viral Hepatitis and Liver Disease, pp. 320-324, herein incorporated by reference in its entirety). HCV-1 core, NS4, and NS5 regions were found to contain serotype-specific epitopes. Individual putative core proteins from HCV-1 and HCV-2 were used as individual antigens to produce antibodies for enzyme-linked immunosorbent assays to detect HCV infection using serologically distinguishable core antigen subtypes (Machida, A. et al. (1992) Hepatology 16:886-891). Simmonds et al. investigated the effect of sequence variability between different types of HCV upon the antigenicity of the NS4 protein by epitope mapping and by enzyme-linked immunosorbent assay (ELISA). These authors mapped two major antigenic regions in the HCV NS4 polyprotein that were recognized by antibody elicited upon natural infection by HCV. Type-specific antibody to particular HCV types was also detected (Simmonds, P. et al. (1993) J. Clin. Microbiol. 31:1493-1503). Ching et al. prepared a series of synthetic peptides based on the sequence of a highly conserved region of the HCV putative nucleocapsid (core) protein and found an immunodominant region that was recognized by human and chimpanzee sera (Ching, W.-M. et al. (1992) Proc. Natl. Acad. Sci. 89:3190-3194).

Assays involving single epitopes as test antigens have the disadvantage that it is difficult to control solid phase coating of the support surface by large numbers of individual epitopes containing short peptides. In such cases where the assay involves deposition of an immunogenic antigen on a solid support, the sensitivity of the assay is limited by the amount of antigen that can be coated on the surface of the solid support.

An example of an immunoassay that includes immunodominant epitopes from different regions of a single virus subtype is disclosed within Chien et al. (Proc. Natl. Acad. Sci. USA 89:10011-10015 (1992), herein incorporated by reference). The assay described by Chien utilizes recombinant HCV polypeptides derived from many different regions of the HCV type 1 polyprotein, including that of chimeric recombinant polyprotein, C25, comprises immunodominant components evident in both the structural and non-structural regions. The polyproteins produced are recombinantly derived viral polypeptides and are included on the surface of an immunoassay in order to capture antibodies, i.e., detect the presence of antibodies generated in response to infection with HCV. However, these polyproteins contain epitopes from a single viral strain thereby limiting the ability to detect anti-HCV antibodies from different strains of the virus.

Claim 1 of 10 Claims

What is claimed is:

1. A method of producing a multiple copy epitope polypeptide useful in an immunoassay for detecting anti-hepatitis C virus (HCV) antibodies comprising the steps of:

(a) identifying nucleotide sequences that encode a plurality of different HCV epitopes;

(b) placing the nucleotide sequences into an expression cassette, wherein said expression cassette encodes a multiple copy epitope sequence comprising the general structural formula (I):

(A)_x--(B)_y --(C)_z (I)

wherein (I) is a linear amino acid sequence;

(B) is an amino acid sequence containing at least five and not more than 1,000 amino acids which amino acids correspond to a naturally occurring antigenic determinant of a hepatitis C virus (HCV) polyprotein;

(A) and (C) are each amino acid sequences different from (B) and different from each other and are each independently an amino acid sequence containing at least five and not more than 1,000 amino acids which amino acids represent an antigenic determinant that is not adjacent to B in naturally-occurring strains of HCV;

x is an integer of 2 or more and at least two (A)s are the same antigenic determinant from the same HCV strain;

y is an integer of 2 or more and at least two (B)s are the same or an equivalent antigenic determinant from different HCV strains; and wherein (A), (B) and (C) are in any linear order;

(c) transforming a suitable host with the cassette in order to express said multiple copy epitope polypeptide; and

(d) purifying the expressed multiple epitope polypeptide, wherein said multiple epitope polypeptide is useful in an anti-HCV immunoassay.
 

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