Title:  Methods for the simultaneous detection of HCV antigens and HCV antibodies

United States Patent:  6,855,809

Issued:  February 15, 2005

Inventors:  Shah; Dinesh O. (Libertyville, IL); Dawson; George J. (Libertyville, IL); Muerhoff; A. Scott (Kenosha, WI); Jiang; Lily (Mundelein, IL); Gutierrez; Robin A. (Gurnee, IL); Leary; Thomas P. (Kenosha, WI); Desai; Suresh (Libertyville, IL); Stewart; James L. (Libertyville, IL)

Assignee:  Abbott Laboratories (Abbott Park, IL)

Appl. No.:  753910

Filed:  January 7, 2004

Abstract

The subject invention relates to methods for the simultaneous detection of Hepatitis C Virus (HCV) antigens as well as antibodies produced in response to HCV antigens. Furthermore, the subject invention allows one to detect antigens in the early, acute stage of infection, even prior to the development of antibodies, thereby allowing for early detection of infected blood and blood products, thus improving the safety of the blood supply.

Description of the Invention

BACKGROUND OF THE INVENTION

1. Technical Field

The subject invention relates to methods for the simultaneous detection of Hepatitis C Virus (HCV) antigens as well as antibodies produced in response to HCV antigens. Furthermore, the subject invention allows one to detect antigens in the early, acute stage of infection, even prior to the development of antibodies, thereby allowing for early detection of infected blood and blood products, and thus improving the safety of the blood supply.

2. Background Information

Recent epidemiological studies indicate that HCV infects more than 170 million people worldwide and that, in more than 50% of the cases, the infection is chronic. In the United States, there are approximately 4 million people infected, and 30,000 new infections are estimated to occur annually (NIH Conference, Hepatology Suppl 1:2S (1997)). In addition, HCV is responsible for 8,000-10,000 deaths annually in the United States and is the leading indicator for liver transplantation.

The HCV genome is a single-stranded RNA molecule of positive polarity that is approximately 9400-9500 nucleotides in length. The organization of the coding regions resembles that of other flaviviruses [Major et al., Hepatology 25:1527 (1997)] as well as the more recently discovered GB viruses [Muerhoff A S, et al., J Virol 69:5621 (1995)]. The HCV genome possesses a large open reading frame (ORF) encoding a polyprotein precursor of 3010 to 3033 amino acids depending on the particular isolate [Choo et al., Proc Natl Acad Sci USA 88:2451 (1991); Grakoui et al., J Virol 67:1385 (1993)]. HCV structural genes (core and envelope) are encoded near the 5'-end of the genome, followed by the proteases and helicase, the helicase cofactor and the replicase. Noncoding regions (NCR), thought to be important in replication, are found at each end of the genome.

HCV infection occurs primarily through parenteral exposure, i.e., through shared needles, by tattooing, or through transfusion of contaminated blood or blood products. Following exposure, the virus enters a susceptible hepatocyte and viral replication occurs. There is an eclipse phase period of approximately 10 days during which time there is no evidence of viral presence (i.e., viral RNA cannot be detected), serum transaminase levels are within normal limits, and there is no evidence of an immune response to HCV [Busch et al., Transfusion 40:143 (2000)]. Typically, about 10 days following exposure, HCV RNA can be detected, often with viral loads between 100,000-120,000,000 HCV RNA copies per ml of serum. Several weeks later, there is typically an increase in ALT levels indicating inflammation of the liver; antibodies are detected an average of about 70 days after exposure.

One of the preventive measures employed to limit the spread of HCV infections is to screen blood for exposure to HCV, either by the detection of antibodies to HCV or by the detection of viral-specific molecules (e.g., HCV RNA or HCV core proteins) in serum/plasma. Blood or blood products derived from individuals identified as having been exposed to HCV, by these tests, are removed from the blood supply and are not utilized for distribution to recipients of blood products (see, e.g., U.S. Pat. No. 6,172,189). These tests may also be utilized in the clinical setting to diagnose liver disease attributable to HCV infection.

Due to the unavailability of native, intact HCV virions, serologic antibody tests have relied on recombinant antigens or synthetic peptides, representing selected fragments of the viral polyprotein. The first generation anti-HCV screening tests were based on detection of antibodies directed against a recombinant protein (HCV genotype 1a) originating from sequences located in the nonstructural NS-4 protein (C100-3) [Choo et al., Science 244:359 (1989); Kuo et al., Science 244:362 (1989)]. The first generation assays failed to detect antibodies in approximately 10% of individuals having chronic HCV infection and up to 10-30% of individuals presenting with acute HCV infection. The second generation anti-HCV assays have incorporated recombinant proteins from three different regions of the HCV genome (HCV genotype 1a), including amino acid sequences from the core, NS3, and NS4 protein [Mimms et al., Lancet 336:1590 (1990); Bresters et al., Vox Sang 62:213 (1992)], allowing a marked improvement over the first generation tests in identifying HCV infected blood donors [Aach et al., N Engl J Med 325:1325 (1991); Kleinman et al., Transfusion 32:805 (1992)]. The second generation assays detect antibodies in close to 100% of chronic HCV cases [Hino K., Intervirology 37:77 (1994)] and in nearly 100% of the acute cases by 12 weeks post infection [Alter et al., N Engl J Med 327:1899 (1992); Bresters et al., Vox Sang 62:213 (1992)]. The third generation test includes a recombinant protein expressing amino acid sequences from the NS5 region, as well as antigens from the core, NS3 and NS4. Some studies have indicated a slight improvement in sensitivity in comparing the third generation tests to second generation tests [Lee et al., Transfusion 35:845 (1995); Courouce et al. Transfusion 34:790-795 (1994)], but this improvement is largely attributed to changes in the NS3 protein rather than the inclusion of NS5 [Courouce et al., Lancet 343:853 (1994)].

In general, the second and third generation HCV antibody tests detect exposure to HCV about 70 days after exposure. Since HCV establishes persistent, and in many cases lifelong infection, the detection of antibodies to HCV represents a very efficient method for determining exposure to HCV. However, antibody testing alone will frequently fail to detect HCV infected individuals during the first 70 days after exposure.

The existing HCV antigen tests rely on detecting the presence of the HCV core antigen in serum or plasma. The core (or nucleocapsid) protein comprises the first 191 amino acids of the polyprotein. Two different types of serologic assays have been developed which permit detection of HCV core antigens in serum. One assay format detects HCV core antigens in subjects prior to seroconversion and is utilized in screening blood donors, while the other assay format detects core antigens only in hepatitis C patients, regardless of their HCV antibody status and is utilized in clinical laboratories to diagnose exposure to HCV or to monitor antiviral therapy.

Recent data on samples obtained during the pre-seroconversion period indicate that the HCV antigen test detects exposure to HCV significantly earlier than antibody testing [Aoyagi et al., J Clin Microbiol 37:1802 (1999); Peterson et al., Vox Sang 78:80(2000); Dawson et al., Transfusion, SD161, 40(2000); Muerhoff et al., 7^th International Meeting on Hepatitis C virus and related viruses, Dec. 3-7, 2000], and represents an alternative to nucleic acid testing for detecting exposure to HCV during the pre-seroconversion period. The advantages of HCV antigen detection are that the test is rapid, simple, may-not require sample extraction or other pretreatment, and is not as prone to handling errors (e.g., contamination) as may occur in the HCV RNA tests.

In clinical laboratories, the HCV antigen test has comparable sensitivity to the HCV DNA tests in detecting exposure to HCV in patients infected with different HCV genotypes [Dickson et al., Transplantation 68:1512 (1999)] and in monitoring antiviral therapy [Tanaka et al., Hepatology 32:388 (2000); Tanaka et al., J Hepatol 23:742 (1995)]. Thus, HCV core antigen tests present a practical alternative to HCV RNA for screening blood donors or for monitoring antiviral therapy.

The uniqueness of the current invention lies in its ability to detect HCV antibodies and HCV antigens simultaneously (see also International Application No. PCT/JP99/04129). This combination test or "combo" assay utilizes antigen detection to identify exposure to HCV during the pre-seroconversion "window period" and antibody detection to identify exposure to HCV after seroconversion.

SUMMARY OF THE INVENTION

The subject invention encompasses a method of simutaneously detecting at least one Hepatitis C Virus (HCV) antigen and at least one HCV antibody in a test sample comprising the steps of: a) contacting the test sample with: 1) at least one HCV viral antigen or portion thereof coated on a solid phase (e.g., a microparticle), for a time and under conditions sufficient for the formation of antibody/antigen complexes and 2) at least one antibody to HCV or portion thereof coated on the solid phase, for a time and under conditions sufficient for the formation of antigen/antibody complexes; b) detecting the antibody/antigen complexes, presence of the complexes indicating presence of at least one HCV antigen in the test sample; and c) detecting the antigen/antibody complexes, presence of the complexes indicating presence of at least one HCV antibody in the test sample. The at least one HCV antigen coated on the solid phase may be, for example, core antigen, NS3, NS4, NS5, and portions (or fragments) thereof. The at least one antibody coated on the solid phase may be, for example, a monoclonal antibody selected from the group consisting of 13-959-270, 14-1269-281, 14-1287-252, 14-153-234, 14-153-462, 14-1705-225, 14-1708-269, 14-1708-403, 14-178-125, 14-188-104, 14-283-112, 14-635-225, 14-726-217, 14-886-216, 14-947-104, 14-945-218, 107-35-54, 110-81-17, 13-975-157, 14-1350-210, C11-3, C11-7, C11-10, C11-14 and C11-15. Further, the at least one monoclonal antibody coated on the solid phase preferably is not reactive with the at least one antigen coated on the solid phase. In particular, the at least one monoclonal antibody may be a HCV anti-core monoclonal antibody and the at least one antigen may be a recombinant HCV core protein. The recombinant core protein does not contain epitopes to which the anti-core monoclonal antibody binds.

Additionally, the present invention includes a method for simultaneously detecting the presence of at least one HCV antigen and at least one HCV antibody in a test sample comprising the steps of: a) contacting the test sample with: 1) at least one HCV viral antigen or portion thereof coated on a solid phase, wherein the solid phase is, for example, a microparticle, for a time and under conditions sufficient for the formation of antibody/antigen complexes and 2) at least one HCV antibody or portion thereof coated on the solid phase, for a time and under conditions sufficient for the formation of antigen/antibody complexes; b) adding a first conjugate to the resulting antibody/antigen complexes for a time and under conditions sufficient to allow the conjugate to bind to the bound antibody in (a) (1), wherein the conjugate comprises a second antibody (e.g., mouse anti-human IgG) attached to a label (for example, a chemiluminescent compound) capable of generating a detectable signal and simultaneously adding a second conjugate to the resulting antigen/antibody complexes for a time and under conditions sufficient to allow said second conjugate to bind to the bound antigen in (a) (2), wherein said second conjugate comprises a third antibody (e.g., a monoclonal antibody to anti-HCV core antigen such as C11-10) attached to the label, for example, chemiluminescent compound, capable of generating a detectable signal; and b) detecting the presence of the generated signal, presence of the signal indicating the presence of at least one HCV antigen or at least one HCV antigen in the test sample. Again, the at least one HCV antigen coated on the solid phase may be selected from the group consisting of core antigen, NS3, NS4, NS5, and portions thereof. Further, the at least one antibody coated on the solid phase may be a monoclonal antibody selected from the group consisting of, for example, 13-959-270, 14-1269-281, 14-1287-252, 14-153-234, 14-153-462, 14-1705-225, 14-1708-269, 14-1708-403, 14-178-125, 14-188-104, 14-283-112, 14-635-225, 14-726-217, 14-886-216, 14-947-104, 14-945-218, 13-975-157, 14-1350-210, 107-35-54, 110-81-17, C11-3, C11-7, C11-10, C11-14 and C11-15.

The at least one monoclonal antibody coated on the solid phase is preferably not reactive with the at least one antigen coated on the solid phase.

Also, the present invention encompasses a kit comprising: a) a container containing at least one HCV antigen coated on a solid phase, wherein the solid phase is, for example, a microparticle; and b) a container containing at least one HCV antibody coated on a solid phase, wherein the solid phase is preferably a microparticle.

The present invention also includes a kit comprising: a container containing: 1) at least one HCV antigen coated on a solid phase, wherein the solid phase is preferably a microparticle, and 2) at least one HCV antibody, coated on the solid phase. The kit may further comprise at least one conjugate comprising a signal-generating compound attached to a HCV antigen or HCV antibody. The signal-generating compound may be, for example, acridinium or an acridinium-containing compound.

Additionally, the present invention includes a method of detecting HCV antigen in a test sample comprising the steps of: a) contacting the test sample with at least one HCV antibody (e.g., monoclonal) coated on a solid phase, wherein the solid phase is a microparticle, for a time and under conditions sufficient for the formation of antibody/antigen complexes; and b) detecting the presence of antibody/antigen complexes, presence of the complexes indicating presence of antigen in the test sample.

The invention also encompasses a method of detecting HCV antigen in a test sample comprising the steps of: a) contacting the test sample with at least one HCV antibody (e.g., monoclonal) coated on a solid phase, wherein the solid phase is, preferably, a microparticle, for a time and under conditions sufficient for the formation of antibody/antigen complexes; b) adding a conjugate to the resulting antibody/antigen complexes for a time and under conditions sufficient to allow the conjugate to bind to the bound at least one antibody, wherein the conjugate comprises a second antibody attached to a label, for example, a chemiluminescent compound capable of generating a detectable signal; and c) detecting the signal generated by the label, for example, chemiluminescent compound, a signal generated by the label indicating the presence of antigen in the test sample.

Also, the present invention includes a recombinant protein comprising an amino acid sequence selected from the group consisting of, for example, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12 and SEQ ID NO:16 as well as an amino acid sequence comprising conservative amino acid substitutions of these sequences. (A conservative substitution is defined as one or more amino acid substitutions in a sequence which do not change the function of the sequence.) The present invention also includes a recombinant protein comprising an amino acid sequence encoded by a nucleotide sequence selected from the group consisting of, for example, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:11 and SEQ ID NO:15.

(Substitutions, deletions and additions within the sequences which do not affect functionally affect the protein encoded by the sequence are also considered to be within the scope of the present invention.)

Additionally, the present invention includes a vector or construct comprising a nucleotide sequence selected from the group consisting of, for example, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:11 and SEQ ID NO:15. The invention also includes a host cell comprising the vector or construct.

Furthermore, the present invention includes an immunoassay which may simultaneously detect at least one HCV antigen or at least one HCV antibody in a test sample.

DETAILED DESCRIPTION OF THE INVENTION

The subject invention relates to various methods which may be utilized in order to simultaneously detect antigens of HCV and antibodies to HCV in a biological sample. Thus, if an individual has either developed specific antibodies to HCV and/or has HCV specific antigens in the biological sample tested, the methods of the present invention will yield a positive result. Such results may be used, for example, to diagnose the patient in terms of presence and status of infection (i.e., acute or chronic) as well as to determine the suitability of a donor blood or blood product sample for transfusion.

Also, the present invention overcomes the problems associated with the "window period" (i.e., 50-60 days post infection) wherein an individual may be infected with HCV but may not have developed antibodies yet. Such individuals may transmit HCV to others during this period. Thus, by detecting HCV during this "window period", the present invention allows for a quick diagnosis of HCV, as opposed to waiting for the development of antibodies, and prevents contamination of the blood supply.

In one embodiment of the present invention, HCV viral antigens (e.g., core, N3, N4 and N5), or portions thereof, are coated on a solid phase (or are in a liquid phase). The test or biological sample (e.g., serum, plasma, urine, etc.) is then contacted with the solid phase. If antibodies are present in the sample, such antibodies bind to the antigens on the solid phase and are then detected by either a direct or indirect method. The direct method comprises simply detecting presence of the complex itself and thus presence of the antibodies. In the indirect method, a conjugate is added to the bound antibody. The conjugate comprises a second antibody, which binds to the first bound antibody, attached to a signal-generating compound or label. Should the second antibody bind to a bound first antibody, the signal-generating compound generates a measurable signal. Such signal then indicates presence of the first antibody in the test sample.

Examples of solid phases used in diagnostic immunoassays are porous and non-porous materials, latex particles, magnetic particles, microparticles (see U.S. Pat. No. 5,705,330), beads, membranes, microtiter wells and plastic tubes. The choice of solid phase material and method of labeling the antigen or antibody present in the conjugate, if desired, are determined based upon desired assay format performance characteristics.

As noted above, the conjugate (or indicator reagent) will comprise an antibody (or perhaps anti-antibody, depending upon the assay), attached to a signal-generating compound or label. This signal-generating compound or "label" is itself detectable or may be reacted with one or more additional compounds to generate a detectable product. Examples of signal-generating compounds include chromogens, radioisotopes (e.g., 125I, 131I, 32P, 3H, 35S and 14C), chemiluminescent compounds (e.g., acridinium), particles (visible or fluorescent), nucleic acids, complexing agents, or catalysts such as enzymes (e.g., alkaline phosphatase, acid phosphatase, horseradish peroxidase, beta-galactosidase and ribonuclease). In the case of enzyme use (e.g., alkaline phosphatase or horseradish peroxidase), addition of a chromo-, fluro-, or lumo-genic substrate results in generation of a detectable signal. Other detection systems such as time-resolved fluorescence, internal-reflection fluorescence, amplification (e.g., polymerase chain reaction) and Raman spectroscopy are also useful.

Examples of biological fluids which may be tested by the above immunoassays include plasma, urine, whole blood, dried whole blood, serum, cerebrospinal fluid, saliva, tears, nasal washes or aqueous extracts of tissues and cells.

At the same time as the antibodies are being detected, HCV antigens are also being detected; thus, the present invention obviates the need for the running of two different tests. This is accomplished by exposing the test sample to a solid phase (or liquid phase) coated with specific antibodies to HCV (e.g., human or animal monoclonal antibodies to core, polyclonal antibodies, chimeric antibodies, etc.). Antigens, if present in the sample, bind to the solid phase and may then be detected by a direct or indirect method as described above. More specifically, the indirect method involves the addition of a conjugate comprising a second antibody (which binds to the bound antigen) attached to a label or signal-generating compound. When the second antibody binds to the bound antigen, a detectable signal is then generated indicating presence of HCV antigen in the test sample.

The antibodies which are coated on the solid phase as well as the "second antibody" may be, as noted above, monoclonal antibodies or polyclonal antibodies. For example, if one chooses to utilize monoclonal antibodies, they may be selected from Abbott monoclonal antibodies 13-959-270, 14-1269-281, 14-1287-252, 14-153-234, 14-153-462, 14-1705-225, 14-1708-269, 14-1708-403, 14-178-125, 14-188-104, 14-283-112, 14-635-225, 14-726-217, 14-886-216, 14-947-104 and 14-945-218. The following anti-core monoclonal antibodies may also be utilized for purposes of the present invention: 107-35-54, 110-81-17, 13-975-157, 14-1350-210 (see U.S. Pat. No. 5,753,430) and Tonen HCV core monoclonals C11-3, 7, 10, 14 and 15 (see PCT Application WO 099/06836), all of which are available from the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209. (For a discussion of the manner in which monoclonal antibodies may be created, see Kohler and Milstein, Nature (1975) 256:494, and reviewed in Monoclonal Hybridoma Antibodies: Techniques and Applications, ed. Hurrell (CRC Press, Inc., 1982); see also J. W. Goding in Monoclonal Antibodies: Principles and Practice (Academic Press, N.Y., 1983; see also U.S. Pat. No. 5,753,430).

It should be noted that HCV core protein may be one possible target of the HCV antigen portion of the assay. More specifically, the detection of the core protein is accomplished by using monoclonal antibodies directed towards epitopes within the core protein. These anti-core monoclonals are placed on the solid phase and facilitate the capture of core antigen proteins from the test sample. For detection of HCV antibodies in the test sample, recombinant HCV core protein is also placed on the solid phase. It should be noted however that there are significant problems associated with the use of a single protein as the target for an antigen test and as the capture reagent for antibody detection, namely there is significant "cross-reactivity" between the core antigen and the anti-core monoclonal antibodies coated onto the solid phase(s). This results in a false positive signal, even in the absence of the test sample, since the monoclonal antibodies will bind to epitopes present on the recombinant protein.

In order to avoid such cross-reactivity, the core protein used in the antibody detection portion of the assay may be modified such that the ability of the anti-core monoclonals to bind HCV core is eliminated. Such modification may be achieved by use of recombinant DNA technology in which the epitope region (i.e., the short sequence of amino acids needed for monoclonal antibody binding) is eliminated or modified. Thus, use of the modified recombinant core protein would consequently maintain several human epitopes to which antibodies present in the serum of infected individuals would bind; however, the anti-core monoclonal antibodies used for antigen capture would not bind the modified protein. Alternatively, one could replace the HCV core recombinant protein with polypeptides that include sequences known to bind to antibodies present in the serum of most infected individuals, but do not include sequences containing the epitopes recognized by the anti-core monoclonals used to detect HCV core antigens.

More specifically, as noted above, in order to avoid cross-reactivity, one may use core antigens for antibody detection in the assay. In particular, in the present invention, the solid phase may be coated with nonstructural proteins (NS) 3, 4 and/or 5 (i.e., NS3, NS4 and/or NS5) and/or the core protein. Alternatively, in the present invention, the solid phase may be coated with any of the above-mentioned HCV proteins, or segments or portions thereof, either individually or in combination (for antibody detection). The antigens used for coating the solid phase may be generated as a contiguous recombinant protein, expressed as recombinant proteins, either as a single entity or as discrete entities, or as synthetic peptides designed either as a single entity or discrete entities.

It should also be noted that one may also detect antibodies to HCV E2 in the combo assay. Thus, using the present assay described herein, one may replace an assay which detects anti-core antibody. Alternatively, one may supplement such an anti-core antibody assay with the antigen assay portion of the combo assay described herein. (See, e.g., U.S. Pat. No. 6,156,495 relating to detection of HGBV E2 antibody or antigen.)

With respect to detection of antigens in the present invention, as noted above, the monoclonal or polyclonal antibodies coated on the solid phase must not recognize the core antigens used on the solid phase (for antibody detection). Thus, for example, in the present invention, one may use the full antibody or a fragment thereof. (For purposes of the present invention, a "fragment" or "portion" of an antibody is defined as a subunit of the antibody which reacts in the same manner, functionally, as the full antibody with respect to binding properties.)

Additionally, it should also be noted that the initial capture antibody (for detecting HCV antigens) used in the immunoassay may be covalently or non-covalently (e.g., ionic, hydrophobic, etc.) attached to the solid phase. Linking agents for covalent attachment are known in the art and may be part of the solid phase or derivatized to it prior to coating.

The second manner in which to use the solid phase for detecting HCV antibodies involves elimination of the core antigens entirely. For example, the solid phase is coated with NS3, NS4 and/or NS5 and a substitute for the core protein or regions thereof (e.g., E2). In contrast, the antibodies coated on the solid phase for detection of antigen are directed against the core protein of HCV.

Other assay formats which may be used for purposes of the present invention, in order to simultaneously detect antigens and antibodies include, for example, Dual assay strip blots, a rapid test, a Western blot, as well as the use of paramagnetic particles in, for example, an Architect.RTM. assay (Frank Quinn, The Immunoassay Handbook, Second edition, edited by David Wild, pages 363-367, 2001). Such formats are known to those of ordinary skill in the art.

It should also be noted that the assays of the present invention may also be used to solely detect HCV antigens or HCV antibodies, rather than both, if desired. Certainly, if one desires to establish that an infection initially exists, one may simply want to determine the presence of antigen in a test sample such as during the "window period". On the other hand, if one wants to establish the stage of infection (e.g., acute versus chronic), one may wish to look for the presence of antibodies and titer thereof.

It should also be noted that the elements of the assay described above are particularly suitable for use in the form of a kit. The kit may also comprise one container such as vial, bottles or strip, with each container with a pre-set solid phase, and other containers containing the respective conjugates. These kits may also contain vials or containers of other reagents needed for performing the assay, such as washing, processing and indicator reagents.

Claim 1 of 4 Claims

What is claimed is:

1. A recombinant protein comprising the amino acid sequence of SEQ ID NO:32.

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