CA2142872A1 - Methods for monitoring effectiveness of interferon therapy in individuals with hcv infections - Google Patents

Abstract

A means for determining the effectiveness of interferon therapy in individuals who are infected with Hepatitis C Virus (HCV) and are receiving such therapy. Monitoring the level of anti-HCV IgM
and/or anti-GOR IgG provide means for establishing whether or not the infected patient is responding to interferon therapy; such monitoring is especially useful for patients diagnosed with chronic acute HCV infections.

Description

2142~72 Wo 94/04922 Pcr/US93/07287 METHODS FOR MONITO~?ING EFFECTIVENESS OF INTERFER(;)N
THERAPY IN INDIYIDUALS WITH HCV INFECTIONS

Back~round of the Invention This invention relates generally to interferon therapy, and more particularly, relates to the monitoring of anti-HCV IgM or anti-GOR
IgG in an indindual's test sample as an indication of the clinical ef~ectiveness and/or clinical outcome of interferon therapy used to treat an individual with chronic HCV infection.
'''' Greater than 90% of cases of transfusion hepatitis worldwide are attributed to non-A, non-B hepatitis (NANBH). The predominant etiological agent of NANBH, termed Hepatitis C virus (HCV), has been ~-cloned. An immunodominant region designated as c-1û0, encoded by 1~ the putative nonstructural (NS)-4 genomic region, has been expressed, purified, and incorporated into immunoassays which are useful in the detection of antibody to HCV in infected test samples. See, for e~ample, Q.-L. Choo et al., Science 244:359-362 (1989); H. J. Alter et al., N. ~n~
J. MedO 321:1494-1500 (1989); J. I. Esteban et al., Lancet ii:294-297 (1989);
G. Huo et al., Science 244:362-364 ~1989); T. Miyamura et al., Proc. Natl.
Acad. Sci~USA 87:983-987 (1990); and C. L. Van der Poel et al., Lancet ii:297-298 (1989).

Recently, Mishiro et al. have reported isolation of a cDNA clone designated as GOR4701, ~om the plasma of a chimpanzee e~perimentally infeted ~th NANBH agent. S. Mishiro et al., ancet 336:1400-1403 (1990). the GOR~7-1 cDNA clone was shown to lack ~^
detectable sequence homology to l~nown HCV sequences. Further, unlike HC~, GOR~gene is clod~d by a single-copy gene of host cellular ` -30 sequence. An ELA has been developed using a 27-amino acid synthetic peptide, spGOR2, deduced from the cDNA sequence of the GOR gene.
Using this EL~. 60% to 80% if the samples were found to be reactive, while only two percent (2%) of the voluntary donor population had anti-GOR2 antibodies, suggesting a st~ong association between immune response to GOR and H~V in NANBH patients.

214287~
wo 94J04922 PCrtUS93/07287 It is known that a percentage of individuals who develop NANBH
(HCV) infections pro~Tess to the chronic state of the disease, where liver function is impaired and death ultimately may result in ~evere cases. Interferon therapy has been shown to improve liver function in a proportion of patients with NANB hepatitis. About fiflGy percent (50%) of chronic Hepatitis C cases respond to alpha interferon (IFN) therapy.
No demographic, biochemical or clinical features have been identified to predict response to therapy. Interferon induced normalization of serum ALT levels is so far the only endpoint indicating response to IFN
10 therapy. Biochemical relapse is predicted by increasing serum ALT
leveis. In addition to ALT elevation, recurrence of HCV viremia as measured by PCR during or after interferon treatment is discussed as a possible predictor for relapse. However, AI,T elevations may not occur for several months after viremia recurrence. It is not clear at this time 1~ if changes în anti-HCV IgG levels are associaterd with IFN treatment.

It would be advantageous to provide a means for predicting the clinical outcome of interferon therapy in an individual receiving such therapy for NANB (HCV) infection. Such a means would be useful in -~

2~ that the response to therapy could be monitored, and the state of disease could be assessed to dete~nine remission or relapse after therapy. ;
Summarv of the Invention The present invention provides a means for determining the ~-~
25 effect*eness and/or possible clinical outcome of interferon therapy when it is used in individuals diagnosed with chronicECV infection, or when interferon therapy is used as a treatment for an HCV infection.
Briefly, test samples from an indindual are taken before the start of interferon therapy and during the course of interferon therapy, and 30 assayed for either anti-HCV IgM and/or anti-GOR IgG antibodies. A
decrease in either a~ti-HCV IgM level or anti-COR IgG level in indicative of a clinical response to interferon therapy. 1-Several assay procedures disclosed herein can be used to assay 35 for anti-HCV IgM activity. HCV antigens useful in these assays include HCV CORE, HCVp33c and HCV c-100. The most preferred antigen is HCV CORE. These assays can include a solid phase to 21 ~28~
:- - WO 94/04922 PCI /US93/07287 which HCV antigen is attached. The solid phase selected can include polymeric or glass beads, nitrocellulose, microparticles, wells of a reaction tray, test tubes and magnetic beads. The signal generating compound can include an enzyme, a luminescent compound, a chromogen, a radioactive element and a chemilllminescent compound.
Examples of enzymes include alkaline phosphatase, horseradish pero~idase and beta-galactosidase. Examples of enhancer compounds include biotin, anti-biotin and avidin. Examples of enhancer compound binding members include biotin, anti-biotin and avidin. In order to 10 block the effects of rheumatoid factor-like substances, the test sample is subjected to conditions sufficient to block the effect of rheumatoid factor-like substance~. These conditions comprise contacting the test sample with a quantity of anti-human IgG to form a mi~ture, and incubating the mi~ture for a time and under conditions sufficient to form a 15 reaction mixture product substantially free of rheumatoid factor-like substance.

Several assay procedures disclosed herein also can be used to ;~
assay for anti-GOR IgG activity. The GOR antigen most preferred in ;~
~) this assay is GOR2. These assays can include a solid phase to which C~OR antigen is attached. The solid phase selected can include polymeric or glass beads, nitrocellulose, microparticles, wells of a - reaction tray, test tubes and magnetic beads. The signal generating compound can include an enzyme, a luminescent compound, a 25 chromogen, a radioactive element and a chemiluminescent compound.
Examples of enzymes include alkaline phosphat~se, horseradish peroxidase and beta-galactosidase. Examples of enhancer compounds include biotin, anti-biotin and avidin. E~amples of enhancer compound bindi~g members include biotin, anti-biotin and avi~in. In order to 30 block the effects of rheumatoid factor-like substances? the test sample is subjected to conditions sufficient to block the effect of rheumatoid factor-like substances. These conditions comprise contacting the test sample with a quantity of anti-human IgG to form a mi~ture, and incubating the mi~ture for a time and under conditions sufficient to form a 35 reaction mi~ture product substantially ~ree of rheumatoid factor-like substance.

wo 94/04~22 ?, 1 ~ ~ 8 7 ~ Pcr/US93/07287 ~-Detailed Description of the Invention The present invention provides a means for determining the effectiveness of interferon therapy for individuals receiving su~h - therapy for HCV infections. The inventions compnses assaying for the 5 presence of anti-GOR IgG and/or anti-HCV IgM and using these determinations to determine clinical effectiveness and/or clinical outcome of an individual.

The present invention employs an immunoassay which utilizes 10 specific binding members. A "specific binding membert" as used herein, is a mem~er of a specific binding pair. That is, two different molecules where one of the molecules through chemical or physical means specifically binds to the second molecule. Therefore, in addition to antigen and antibody specific binding pairs of common ~5 immunoassays, other specific binding pairs can include biotin and avidin, carbohydrates and lectins, complementary nucleotide sequences, effector and receptor molecules, co~actors a~d enzymes, enzyme inhibitors and enzymes, and the like. Furthermore, specific binding pairs can include members that are analogs of the original a~ specific binding members, for e~ample, an analyte-analog.
Immunoreact*e specific binding members include antigens, antigen fragments, antibodies and antibody fragments, both monoclonal and polyclonal, and complexes thereof, including those formed by recombinant DNA molecules. The term "hapten", as used herein, 25 refers to a par~al antigen or non-protein binLing member wh~ch is capable of bir}ding to an antibody, but which is not capable of eliciting antibody formatio~ unless coupled to a carTier protein.

A "capture reagent", as used herein, refers to an unlabeled 30 specific binding member which is specific either for ~he analyte as in a sandwich assay, for the indicator reagent or analyte as in a competitive assay, or ~or an ancillary specific binding member, which itself is specific for the analyte, as in an indirect assay. The capture reagent can be directly or indirectly bound to a solid phase material before the 35 per~ormance of the assay or during the performance of the assay, thereby enabling the separa~on of immobilized complexes from the test sample.

21~.2~7~
. WC) 94/04922 PCr/US93/07287 Test samples which can be tested by the methods of the present inven~on described herein include human and animal body fluids such ;
- as whole blood, serum, plasma, cerebrospinal fluid, urine, biological 5 fluids such as cell culture supernatants, tissue specimens and cell specimens.

An enhancer can be used to detect the generated signal in the assay. By "enhancer" is meant a moiety which can bolster a signal 10 generated in an immunoassay, thereby amplifying the generated signal. Several me~ods of enhancing and amplifying a signal generated in an immunoassay are known in the artO Also, the use of a signal enhancer such as the use of avidin-biot;in also is known. For example, U. S. Patent No. 4,228,237 to Hevey et al. describes the use of a 15 biotin labelled specific binding substance for a ligand used in a method which also employs an enzyme labelled with avidin. The use of a biotin-anti-biotin system is described in European Patent Application No.
160,900, published on November I3, 1985.

ao The term "probe," as used herein, means a member of the specific binding pair attached to an "enhancer" compound. An "enhancer" compound can be any compound used in the assay which can enhance the signal generated by the ~ignal generating compound.
Thus, enhancer compounds include haptens such as biotin, and also include fluorescein, di-nitrophenol, and the like.

The indicator reagent comprises a signal generating compound (label) which is capable of generating a measurable signal detec~able by e~ternal means conjugated (attached) to a specific ~inding member.
"Specific binding member,~ as used herein, means ~l member of a - ~pecific binding pair. That is, two different molecules where one of the - molecules through chemical or physical means specifically binds to the second molecule. In addition to being an antibody member of a specific binding pair for HCV, the indicator reagent also ca~ be a member of 35 any specif ic binding pair, including either hapten-anti-hapten systems such as biotin or anti-biotin, avidin or biotin, a carbohydrate or a lectin, a complementary nucleotide sequence, an effector or a receptor 2142~72 WO ~4/04922 PCI /US~3/072~7 ' molecule, an enzyme cofactor and an enzyme, an enzyme inhibitor or an enzyme, and the like. An immunoreactive specific binding member can be an antibody, an antigen, or an antibody/antigen comple~ that is capable of binding either to HCV as in a sandwich assay, to the capture 5 reagent as in a competitive assay, or to the ancillary specific binding member as in an indirect assay. Thus, if an enhancer is utilized in the assay, the indicator reagent comprises a signal generating compound conjugated to an enhancer-specific compound (enhancer compound binding mem~er), such as biotin or anti-biotin, avidin or biotin, and 10 others known to those skilled in the art. For example, if the enhancer compound utilized is biotin, then anti-biotin, or avidin, can be used as the enhancer-specific compound.

The various signal generating compounds (labels) contemplated ~5 include a chromogen such as bromo-chloro-indole-phosphate (BCIP), catalysts such as enzymes, luminescent compounds such as fluorescein and rhodamine, chemiluminescent compounds such as acridinium, phenanthridinium or 1,2-dio~etane compounds, radioactive elements, and direct visual labels. Egamples of enzymes 2~ include alkaline phosphatase, horseradish pero~idase, beta-galactosidase, and the like. The selection of a particular label is not critical, but it will be capable of producing a signal either by itself or in conjunction with one or more additional substances, such as the use of enzyme substrates when enzymes are employed as the signal 25 generating compound.

It is contemplated that the reagent employed for the assay can be provided in the form of a kit with one or more containers such as vials or bottles, with each container containing a separate reagent such as a 30 monoclonal antibody, or a cocktail of monoclonal antibodies, employed - in the assay.

The assay co~figuration may involve the use of a solid phase in performance of the present invention. A "solid phase", as used herein, 35 refers to any material which is insoluble, or can be made insoluble by a subsequent reaction. The solid phase can be chosen for its intrinsic ability to attract and immobilize the capture reagent. Alternatively, the 2~ ~2C7,~ ~
: wo 94/04922 Pcr/US93/07287 solid phase can retain an additional receptor which has the ability to attract and immobilize the capture reagent. The additional receptor can include a charged substance that is oppositely charged wit~h respect - to the capture reagent itself or to a charged substance conjugated to the 6 capture reagent. As yet another alternative, the receptor molecule can be any specific binding member which is immobilized upon the solid phase and which has the ability to immobilize the capture reagent through a specific binding reaction. The receptor molecule enables the indirect binding of the capture reagent to a solid phase material before -the performance of the assay or during the performance of the assay. If an assay device is utilized to perform the assays of the present invention, it can have many configurations, several of which are dependent upon the material chosen as the solid phase. For example, the solid phase can include any suitable porous material. By "porous ~5 is meant that the material is one through which the test sample caneasily pass and includes both bibulous and non-bibulous solid phase materials. In the present invention, the solid phase can include a fiberglass, cellulose, or nylon pad for use in a pour and flow-through assay device having one or more layers containing one or more of the 2D assay reagents; a dipstick for a dip and read assay; a test strip for wicking (e.g., paper) or thin layer chromatographic or capillary action (e.g., Mtrocellulose) techniques; or other porous or open pore matenals well known to those skilled in the art (e.g., polyethylene sheet material).
The solid phase, however, is not limited to porous materials. The solid phase can also comprise polymeric or glass beads, microparticles, tubes, sheets, plates, slides, wells, tapes, test tubes, or the like, or any other material which has an intrinsic charge or which can retain a charged substance.

.
Natural, synthetic, or naturally occurring materials that are synthetically modified, can be used as a solid phase includi~g polysaccharides, e.g., cellulose materials such as paper and cellulose derivatives such as cellulose acetate and nitrocellulose; silica;
inorganic materials such as dea~tivated alumina, diatomaceous earth, MgSO4, or other inorganic finely divided mate~ial uniformly dispersed in a porous polymer matri~, with polymers such as vinyl chloride, vinyl chloride-propylene copolymer, and vinyl chloride-vinyl acetate 21~287 ?
WO 94/04922 ; ~ PCr/VS93/07287~ -copolymer; cloth, both naturally occurring (e.g., cotton) and synthetic (e.g., nylon); porous gels such as silica gel, agarose, dextran, and gelatin; polymeric films such as polyacrilamide; and the like. ~The solid phase should have reasonable strength or strength can be provided by 5 means of a support, and it should not interfere with the production of a detectable signal.

Preferred solid phase materials for flow-through assay devices include filter paper such as a porous fiberglass material or other fiber 10 matri~ materials. The thickness of such material is not critical, and will be a matter of choice, largely based upon the properties of the s~mple or analyte being assayed, such as the fluidity of the test sample.

To change or enhance the intrinsic charge of the solid phase, a L~ charged substance can be coated directly to the mateI~al or onto microparticles which are then retained by a solid phase support material. Alternatively, microparticles can serve as the solid phase, by being retained in a column or being suspended in the mixture of soluble reagents and test s~mple, or the particles themselves can be retained ao and immobilized by a solid phase support material. By "retained and immobilized" is meant that the particles on or in the support material are not capable of substantial movement to positions elsewhere within ~e support material. The particles can be selected by one skilled in the art from a~y suitable type of particulate material and include those 25 composed of polystyrene, polym~thylacrylate, polypropylene, late~, polytetrafluoroethylene, polyacrylonitrile, polycarbonate, or similar materials. The size of the particles is not critical, although it is preferred that the average diameter of the particles lbe smaller than the average pore size of the support material being used.
Solid supports are known to those in the art and include the walls of wells of a reaction tray, test tubes, polystyrene beads, magnetic beads, nitrocellulose strips, membranes, microparticles such as late~
par~cles, chips of glass, plastic, derivatized plastic, metal and silicon, 35 and others.

21~2Q7~

9 ,;
Accordingly, a test sample which may contain HCV IgM is contacted with a solid support to which HCV antigen has been attached, to form a mixture. This mixture is incubated for a time and under conditions sufficient to form HCV antigen/antibody complexes.
5 Then, a probe comprising a mammalian anti-human IgM to which an enhancer has b~en attached is contacted with the HCV
antigen/antibody complexes, to form a second mixture. This second mi~ture is incubated for a time and under conditions sufficient to form a second mixture reaction product. Ne~t, an indicator reagent which 10 comprises an enhancer compound binding member and a signal generating compound capable of generating a measurable signal is contacted with the second mixture reaction product . This third mi~ture is incubated for a time and under conditions sufficient to form ~ ?
indicator reagent reaction products. The presence and/or amount ~of 15 HCV IgM is determined by detecting the signal generated. The amount of HCV IgM present in the test sample is proportional to the signal generated.

Another assay comprises an assay wherein a test sample which ao may contain HCV IgM is contacted with a solid support to which HCV
an~igen has been attached, to form a mixture. This mi~ture is ~-incubated for a time and under conditions sufficient to form HCV
antigen/antibody complexes. Then, an indicator reagent which comprises a signal generating compound capable of generating a ;
2B measurable signal attached to a specific binding member for HCV IgM
is contacted with the complexes, to form a second mixture. This second m~ture is incubated for a time and under conditions sufficient to form a reaction. The presence and/or amount of HCV IgM present in the t~st sample is deter~mined by detecting the signal genl~rated. The 30 amount of HCV IgM present in the test sample is proportional to the signal generated.

In yet another assay configuration, a test sample is contacted with mammalian anti-human IgM which is coated on a solid phase, 35 and reacted for a time and under conditions sufficient for human IgM/a~ti-human IgM comple~es to form. These comple~es then are contacted with a probe which comprises at least one HC~T antigen 21~2872 WO 94/04922 PCrlUS93/07287 '.

selected from HCV CORE, HCV 33c and HCV c-100 attached to an enhancer compound. The prefelTed enhancer compound is biotin.
These are rPacted for a time and under conditions sufficient to form antigen/antibody/antibody complexes. Ne~:t, these complexes are 5 contacted with an indicator reagent comprising a signal generating compound conjugated to an enhancer compound bindi~g member. The most preferred signal generating compound is the enzyme alkaline phosphatase. The most preferred enhancer compound binding member is anti-biotin. The resultant mixture is reacted for a time and 10 under conditions sufficient for a reaction to occur. If an enzyme is utilized, the signal is detected and measured after addition of an enzyme substrate. The amount of HCV IgM present in the test sample is proportional to the signal generated.

~6 Yet other assay confi~rations can be adapted to detect HCV IgM
by practicing the teachings of this invention, and are contemplated to be within the scope of this invention The presence of anti-GOR IgG antibody also can be assayed and 20 monitored during and after interferon therapy for HCV infection by using the assays disclosed hereinabove and substituting the appropriate GOR antigens. An antibody test for GOR peptides utilizing the synthetic spGOR2 and spGOR346 peptides is known in the art and disclosed in S. U. Mehta et al., J. Clin. Immunolo~r 12(3):178-184 (1992).
25 Thus, a test sample which may contain GOR IgG is contacted with a solid support to which GOR antigen has been attached, to form a mi~ture. This mi~ture is incubated for a time and ulnder conditions sufficient to form GOR antigen/antibody comple~es. Then, a probe comprising a mammalian ajnti-human IgG to which an enhancer has 30 been attached is contacted with the GOR antigen/antibody comple~es, to form a second mi~ture . This second mi~ture is incubated for a time and under conditions sufficient to form a second miYture reaction product. Ne~Rt, an indicator reagent wbich comprises an enhancer compound binding member and a signal generating compound capable 35 of generating a measurable signal is contacted with the second mi~cture reaction product . This third mi~ture is incubated for a time and under conditions sufficient to form indicator reagent reaction products. The 2142~7~ `
`- W 0 94/04922 PC~r/US93/07287 presence and/or amount of GOR IgG is determined by detecting the signal generated. The amount of GOR IgG present in the test sample is proportional to the signal generated.

Another assay comprises an assay wherein a test sample which may contain GOR IgG is contacted with a solid support to which GOR
antigen has been attached~ to form a mixture. This mi~ture is incubated for a time and under conditions sufficient to form GOR
antigen/antibody complexes. Then, an indicator reagent which comprises a signal generating compound capable of generating a measurable signal attached to a specific binding member ~or GOR IgG
is contacted with the complexes, to form a second mixture. This second mixture is incubated for a time and under conditions sufficient to form a reaction. The presence and/or amount of GOR IgG present in the test sample is determined by detecting the signal generated. The amount of GOR IgG present in the test sample is proportional to the signal generated.

In yet another assay configuration, a test sample is contacted ao with mammalian anti-human IgG which is coated on a solid phase,and reacted for a time and under conditions sufficient for human IgG/anti-human IgG complexes to form. These complexes then are contacted with a probe which comprises at least one GOR antigen selected from spGOR346 and spGOR2 attached to an enhancer compound. The preferred enhancer compound is biotin. These are reacted for a time and under conditions suf~cient to form antigen/antibody/antibody comple~es. Next, these complexes are contacted with an indicator reagent comprising a signal generating compound conjugated to an enhancer compound bL~lding member. The most preferred signal generating compound is the lenzyme alkaline phosphatase. The most preferred enhancer compound binding - member is anti-biotin. The resultant mixture is reacted for a time and under conditions sufficient for a reaction to occur. If an enzyme is u~ilized, the signal is detected and measured afl;er addition of an enzyme subs~rate. The amount of GOR IgG present in the test sample is proportional to the signal generated.

21~287 ~
W~ 94/04922 PCI /US93/07287 (: -.

Yet other assay configurations can be adapted to detect GOR IgG
by practicing the teachings of this invention, and are contemplated to be within the scope of this invention. ~ ~
. .
When assaying for HCV antl-IgM, it is preferred that the test sample be treated such as to remove rheumatoid factor-like substances which may be present in the test sample and which may interfere with the per~ormance of the assay. Such treatment can be performed in a variety of ways known to those skilled in the art and include preadsorbing the test sample with protein A or protein G, heat aggregated IgG, and the subjection of the test s~mple to an amount of anti-human IgG sufficient to bind a substantial amount of the interfering rheumatoid factor-like substances. The most preferred me'~od for treating the test sample comprises diluting the test sample 1~ in a diluent sample buffer which contains an amount of goat anti-human IgG suffirient to bind the rheumatoid factor-like substances which may be present in the test sample. This dilution step preferably is performed prior to contacting the test sample with the capture reagent, HCV antigen. The preferred buffer is one which can remove ao any interfering IgG which may be present in the test sample. Thus, buffers which contain a sufficient quantity of anti-IgG can be used as the diluent sample buffer. E2~amples Qf buffers that can b~ used in the assay include Tris buffered saline, phosphate buffered saline, and others known to those skilled in the art. The most preferred buffer comprises a Tris buffered saline (pH 7.2) to which goat anti-human IgG
has been added. Fur~her, other compounds may be added to this buf~er to block non-specific binding. The selection of these compolmds depends upon the constituents chosen for the assay, and are within the ordinar~ skill of the a~rtisa~.
The oIigin of the mammalian anti-human IgM or anti-IgG may be goat, rabbit, sheep, or other mammalian anti-human IgM known in the art. Preferably, the m~mmalian origin of the anti-human IgM and anti-human IgG is goat.
When HCV or GOR antigen is used as a capture reagent in the assays described herein, at least one HCV or GOR antigen is used, 2~ ~2872 :
W O 94/04922 P ~ /US93/07287 either when attached to a solid phase or in solution These HCV
antigens include HCV CORE, HCV 33c and HCV c-100. The GOR
antigens include GOR2 and GOR 346. We have determined th~t HCV
CORE is the most preferred antigen to utilize in performing the assay - 5 for detection of anti-HCV IgM antibodies, but that HC~ 33c and HCV
c-100 also can be used alone or in any combination. Thus, HCV CORE
antigen can be combined with HCV 33c and/or c-100, or other HCV
antigens, and used as capture antigens in methods described herein.

It ~lso is contemplated that a sandwich assay can be performed wherein a soluble capture reagent can include an analyte-specific binding member which has been ~ound to ~ charged substance such as an anionic substance. The present invention also can be used to conduct a competitive assay. In a competitive configuration~ the soluble 1~ capture reagent again includes a specific binding member which has been attached to a charged substance, such as an arlionic polymer, with which to bind a specific binding partner.

Alterna~vely, it also is contemplated that the assay can be performed by scanning probe microscopy, in which an analyte, analyte analog or analyte specific substance which has been bound to a test piece, is contacted with the test sample suspected of containing the analyte, incubated for a time and under conditions suf~cient for a reac~on to occur, and then the presence of analyte is determined by using scanning probe microscopy.

The present invention will now be described by way of Examples, which are intended to demonstrate, but not to limit, the spirit a~d scope of the in~ention.
EXAMPLES
- Example 1 Anti-IgM Activitv 1. Patient Selection and CateFQriza~ioIl Thirty-four (34) patients with chronic active hepatitis C at di~erent time points during interferon therapy (1 to 5 million units of interferon for at least si2~ (6) months) were included. Complete responders (CR) (11/34) were defined by normaliza~on of serum 21~2S,~7 `,~
wo 94/04922 PCr/US93/07287 ' transaminases during treatment. Non-responders (NR) (23/34) were defined as those individuals who showed transaminases levels above two (2) times the normal values (28 IU/MI.). In total, 119 s~mples were tested for HCV IgM antibodies. Samples were collected appro2~imately one (1) month before, two (2) months after the beginning of and at the completion of IFN therapy. In a number of patients, samples drawn approximately six (6) months after therapy also were available. -2. EICV Detection in Samples All samples initially were tested by the Abbott HCV 2nd 10 Generation assay ELA and the Abbott HCV Supplemental EIA
(available ~om Abbott Laboratories, Abbott Park, IL and Abbott, GmbH
Diagnostika, Weisbaden, Germany). Anti-HCV IgM was determined by utilizing a solid phase enzyme immunoassay ("IgM Combo", available from Abbott, GmbH Diagnostika, Weisbaden, Germany). The 1~ anti-HCV IgM assay utilized recombinant antigens derived from the structural (core) and non-structural (NS3/NS4) part o~ the virus. These methods are detailed in (~hau et al., ~. Virol. Methods. 35:343-352, 1991).
The HCV antigens included clone c100-3 (as described by Kuo et al., `~
Science 244:362-364 ~1989]) chimeric polypeptide expressed in yeast, plus ao recombinant HCV polypeptides expressed in E~coli that included those from pHCV-23 (c100 fragment, lacking the lSrst 107 N-terminal amino acids)~ pHCV-29 (CKS-33c), pHCV-34 (CKS-CORE) and pHCV-35 (~ pI.
CORE), and pHCV-45 (NS4/NS5 junction). The amino acids sequences of these HCV polypep~des are known in the art and are described in 25 European Patent Application 0 388 232, published September 19, 1990 (see pages 32 and 34).
All HCV proteins were expressed as CMP-KDO synthetase (CKS) fusion proteins (as taught by T. J. Bolling and W. Mandecki, "An ~s~herichia coli expression vector for high-level production of 30 heterologous proteins in fusion with CMP-KDO synthetase,"
Biotechniaues 8:488-490 [1990]), with the egception of pHCV-35, which was egpressed in a lambda (O pL expression system.
Anti-HCV IgM res~lts were egpressed as specimen absorbance divided by mean absorbance (both absorbance measured at 492 nm) of 35 three (3) negative controls (S/N). In 45~ healthy blood donors, the mean S/N value was 1.0 i 0.5 (mean S/N + 1 standard deviation [SDJ).
Samples with S/N > 4 were characterized further for their IgM antigen 2142~72 `- W094/04922 PCI/US93/~7287 specificity using single bead HCV IgM assays utilizing the HCV-Core, HCV-NS3 and HCV-NS4 antigens.
A neutralization assay using a different recombinant copstruct was used for confirmation testing. Rheumatoid factor interference was 6 eliminated by modifying the specimen diluent as taught hereinabove, which allowed the separation of IgG antibodies.

3. Results Before IFN therapy anti-HCV IgM values were d~termined to be significantly higher in NR than in CR ~t-test). In contrast, no significant di~erence was seen for serum AI,T levels in NR and CR
before IFN therapy (t-test). In patients with complete response to I~N
therapy, decreasses of HCV IgM and ALT levels be~ore, during and after therapy was observed. In patients who e~ibited no response to IFN therapy, HCV IgM and ALT levels both remained elevated.
Nineteen of twenty-three (83%) patients of the NR group had high or increasing HCV IgM levels throughout IFN therapy. It also was observed that in both the NR and CR anti-H(~7 IgM response was directed almost always against the structural (Core) p~t of the virus. -~
Low level anti-C100 or anti-33c IgM was found only in 1/34 patients ao tested. These data are summarized in the following Table 1 which summarizes HCV IgM values (S/N) and ALT levels seen in chronic hepatitis C Patients with no response, and in Table 2 which summarizes HCV IgM values (S/N) and ALT levels seen in chronic hepatitis C Patients with complete response ~
Table 1 (No Response 1;Q IFN~Q, Pa~ent~ - 23 ) Before IFN At start of IFN At enc! of IFN After IFN
Theraov Thera~v I h~ Thera~
Mean IgM 13.9 + 13.8 13.5 + 13.7 13.8 ~ 13.8 16.8 + 13.8 (SIN) +SD
No. Samples 23 ~2 ~ 13 Mean ALT 88+35 66+25 81+58 86+42 (IUI~IL) +SD
No. Samples 16 ~ ~ ao 21l~2~7 `2 wo 94/04922 PC~/US93/07287 Table 2 (ComplQte Res~onse to EN~ No. Patients _ 11) Before IFNAt~ tofIFN Atend of IFN After IFN
Thera~v Thera~Y TheraDv Thera~v Mean IgM6.3 + 6.0 4.4 + 3.4 3.6 + 4.3 2.2 + 1.4 (S/N) +SD
No. Samples 10 11 11 7 Me~n ALT 130+163 18+13 15+14 15+1Q
(IU/ML) +SD
No. Sampl~s 8 11 10 8 5 4. Conclusions From the data presented herein, anti-HCV IgM ~core) levels can predict response to IF therapy. It also was observed that decreasing an~-HCv IgM (core) levels can be an early indicator for relapse du~ing and after IFN therapy. Also, it was observed that increasing anti-H(~v 10 IgM (core) levels can be an early indicator for relapse during and after IFN therapy. Finally, anti-HCv IgM can give additional diagnostic information in conjuction with ALT serum levels for patients with chronic hepatitis C infections.

L~ E}~ampl~ 2 Anti-GOR I~G ~c~i~tv 1. Patient Selection~.nd (~at~ori~ion Thirty-three (33) patients with chronic active hepatitis C at different time points dunng interferon therapy (1 to 5 million units of ao interferon alpha-2B, three times a week, for at least six (6) months) were included. Complete responders (CR) (11/33) were defined by normalization of serum transaminases during treatment. Non-responders (NR) (22/33) were defiIled as those individuals who showed transaminases levels above two (2) times the normal values (28 IU/M~).
25 SamI)les were tested for GOR IgG and GOR IgM antibodies. Samples we~e collected appro2~mately one (1) month before, two (2) months after the beginning of and at the completion of IFN therapy. In a number of patients, samples drawn approgimately six (6) months after therapy also were available.

2142~7~
~ w0 94/04922 Pcr/US93/07~87 2. GOR Detection in Samvles All samples initially were te~ted by the Abbott ~CV 2nd ~-eneration assay ELA and the Abbott HCV Supplemental ELA
(available from Abbott Laboratories, Abbott Park, IL and Abbott, GmbH
Diagnostika, Weisbaden, Germany). Anti-HCV IgM was determined by utilizing a solid phase enzyme immunoassay ("IgM Combo", available from Abbott, GmbH Diagnostika, Weisbaden, Germany). The anti-HCV IgM assay utilized recombinant antigens derived from the structural (core) and non-structural (NS3/NS4) part of the virus. These methods are detailed in Chau et al., J. Virol. Methods. 35:343-352, 1991).
The HCV antigens included clone c100-3 (as described by Kuo et al., Science 244:362-364 [1989]) chimeric polypeptide expressed in yeast, plus recombinant HCV polypeptides expressed in E. coli that included those from pHC~1-23 (c100 fragment, lacking the first 107 N^terminal amino L5 acids), pHCV-29 (CKS-33c), pHCV-34 (CKS-CORE) and pHCV-35 (~ pL
CORE), and pHCV-46 (NS4~NS5 junction). The amino acids sequences of these HCV polypeptides are known in the art and are described in European Patent Application 0 388 232, published September 19, 1990 (see pages 32 and 34).
All HCV proteins were expressed as CMP-KDO synthetase (CKS) fusion proteins (as taught by T. J. Bolling and W. Mandecki, "An Escherichia coli expression vector for high-level production of heterologous proteins in fusion with CMP-KDO synthetase,"
BiotechIi~ues 8:488-490 [1990]), with the exception of pHCV-35, which was expressed in a lambda (O pL expression system.
Anti-GOR IgG and IgM levels were determied by the assay method for IgG described hereinabove and disclosed in S. U. Mehta et al., J. Clin. Immunol. 12 ~3):178-183 (1992). VVhen assaying for aIlti-GOR IgM levels, t~e appro~riate rare reagents were those as descriibed for the HCV IGM assay, but utilizing the synthetic peptides spGOR346 and spGOR2.
3. Results It was found that 11 patients had normalized ALT levels at the end of ~erapy (CR). Non-responders (NR) numbered 22. It was 35 observed that eight of eleven (73%) of CR showed decrease of anti-GOR
IgG. Further, anti-GOR IgM was higher in NR than CR before 2142~7`~
wo 94/04922 Pcr/~lss3/o7287 therapy, while there were no significant changes in this value during therapy.

It thus is possible to monitor the ef~ectivness of interferon 5 therapy during its use in individuals infected with HCV, especially those who have been diagnosed with chronic acute HCV infection. The ~mount of anti-HCV IgM pre-interferon therapy and/or anti-GOR IgG
pre-interferon therapy is compared to the amount of of anti-HCV IgM
pre-interferon therapy and/or anti-GOR IgG pre-interferon therapy 10 during therapy. A decrease in the level of HCY IgM antibodies or anti-GOR IgG antibodies during therapy is an indication that the patient is responding to the interferon therapy. Also, it is possible that dosages may be varied depending upon the results obtained by this monitoring.
The embodiments described and presented herein are intended as ~5 examples rather than as limitations. Thus, the descr~ption of the invention is not intended to limit the invention to the par~cular embodiments disclosed, but it is intended to encompass all equivalents and subject matter within the spirit and scope of the invention as described and contemplated above, and as set forth in the following a~ claims.

Claims (4)

WHAT IS CLAIMED IS:

1. An method for determining the effectiveness of interferon therapy in an individual infected with HCV, comprising:
(a) determining the pre-interferon amount of anti-HCV IgM
or anti-GOR IgG in a test sample of a patient infected with HCV;
(b) determining the amount of anti-HCV IgM and/or anti-GOR IgG in an individual during inteferon therapy by assaying for anti-HCV IgM or anti-GOR IgG in at least one other test sample;
(c) comparing the pre-interferon amount of anti-HCV IgM or anti-GOR IgG with the amount of anti-HCV IgM or anti-GOR IgG
present in a different test sample obtained from the individual during interferon therapy;
wherein a decrease in anti-GOR IgG and/or anti-HCV IgM
indicates complete response in said individual.

2. The method of claim 1, wherein the GOR antigen is selected from the group consisting of GOR2 and GOR346.

3. The method of claim 1, wherein the HCV antigen is selected from the group consisting of HCV-core, HCV-33c and HCV-c-100.

4. The method of claim 1 wherein said HCV antigen is HCV-core.