IgM-antibody response to hepatitis C virus antigens in acute and chronic post-transfusion non-A, non-B hepatitis

Journal of Virological Methods, 35 (1991) 343-352 0 1991 Elsevier Science Publishers B.V. / All rights reserved / 0166-0934/91/%03.50

343

VIRMET 01264

IgM-antibody response to hepatitis C virus antigens in acute and chronic post-transfusion non-A, non-B hepatitis Kurt H. Chau’, George J. Dawson’, Isa K. Mushahwar’, Robin A. Gutierrez’, Rhonda G. Johnson’, Richard R. Lesniewski’, Lars Mattsson2 and Ola Weiland2 ‘Experimental Biology Research, Abbott Laboratories, North Chicago, IL, U.S.A. and ‘Department of Infectious Diseases, Karolinska Institute, Roslagstull Hospital, Stockholm, Sweden

(Accepted

10 August 1991)

Summary

A specific IgM solid-phase enzyme-linked immunoassay for the diagnosis of a recent infection by hepatitis C virus (HCV) was developed. The assay utilizes a structural antigen encoded by sequences at the 5’ end of HCV (core region) and non-structural (NS) antigens encoded by the NS-3 (33~) and NS-4 (~100-3) regions of the HCV genome. Serial serum samples from several clinically diagnosed post-transfusion non-A, non-B hepatitis patients were analyzed for anti-HCV IgM. This antibody was frequently but transiently detected. AntiHCV core IgM was more frequently detected than anti-clOO-3 or anti-33c IgM. In individuals who resolved their HCV infection or progressed to chronicity, anti-HCV IgM was produced transiently at or near the onset of clinically diagnosed acute hepatitis. Anti-HCV IgG; Anti-HCV IgM; Post-transfusion

hepatitis

Introduction

In the past, non-A, non-B hepatitis (NANB-H) has been diagnosed by the exclusion of serodiagnostic markers of hepatitis A, hepatitis B, Epstein Barr Correspondence

to:

Chicago, IL, U.S.A.

I.K. Mushahwar,

Experimental

Biology Research, Abbott Laboratories,

North

344

and cytomegalo viruses. Recently, hepatitis C virus (HCV), the major agent responsible for parenterally transmitted NANB-H, was concentrated from the plasma of a chimpanzee with NANB-H, and segments of the viral genome were cloned and expressed as recombinant antigens (Kuo et al., 1989; Choo et al., 1989). Using recombinant antigens, immunoassays for the detection of antiHCV IgG were developed (Kuo et al., 1989). Anti-HCV IgG is detected usually within 6 weeks after infection and hence is used as a screening rather than as a diagnostic test for recently acquired HCV infection (Mattsson et al., 1991). Virus specific IgM-class antibody, is an accurate serologic marker for the diagnosis of a recent viral infection. It has been routinely utilized for this purpose in rubella (Meurman et al., 1977), cytomegalovirus (Kangro, 1980), hepatitis A (Flehmig et al., 1979), and hepatitis B (Chau et al., 1983) viral infections. In this report we describe the result of the detection of anti-HCV IgM in serum specimens obtained from different categories of donors and clinically diagnosed HCV cases.

Materials and Methods Blood donors and patients Sera from 392 healthy volunteer blood donors and 247 ALT-elevated plasmapheresis donors were obtained from South East Wisconsin Blood Center (Milwaukee,.WI) and Pyramid Biologicals (Van Nuys, CA), respectively. Serial serum samples were obtained from 3 plasmapheresis donors who were routinely monitored by a local blood center for ALT levels and antibodies to HBV, HCV and HIV. Sera from 5 patients with confirmed post-transfusion NANBH (PT-NANBH) were obtained from Roslagstull Hospital (Stockholm, Sweden). Sera from patients diagnosed with acute hepatitis A, acute hepatitis B, chronic hepatitis B and PT-NANBH infections, and sera from hemodialysis patients were withdrawn from pedigreed in-house stored panels donated by a variety of U.S. and European collaborators. HCV antigens The HCV ~100-3 antigen expressed in S. cerevisiae as a fusion protein with superoxide dismutase was obtained from Chiron Corporation (Emeryville, CA). A second recombinant antigen designated as clone 33c in the European patent filed by Chiron Corporation, and a third recombinant antigen derived from a region believed to encode for the core protein (Desai et al., 1991), were cloned and expressed at Abbott Laboratories as chimeric fusion proteins with E. coli CMP-KDO synthetase (CKS). A synthetic peptide prepared by a standard solid-phase synthesis procedure (Barany and Merrifield, 1980) representing the core region (~~75) was also used in a competitive confirmatory procedure as described below.

345

Solid-phase HC V antigens Polystyrene beads (6 mm in diameter) were coated with purified HCV antigen(s) preparation (0.5 pg/ml) in 0.1 M phosphate buffered saline (pH 7.4) for 2 h at 40°C. ELISA for anti-HCV ZgG and anti-HCV IgM Anti-HCV IgG Anti-HCV IgG assays utilizing a variety of HCV antigens as solid phases were performed as described previously (Dawson et al., 1991). Anti-HCV IgM Serum anti-HCV IgM was determined by a solid-phase assay. The sample to be tested was diluted 1441 in a sample diluent (Dawson et al., 1991) containing 2% goat anti-human IgG (g). The diluted sample (0.2 ml) was incubated for 2.5 h at 40°C with the HCV-coated bead; after washing with water, the bead was further incubated with 0.2 ml of goat anti-human IgM-HRP conjugate (Kirkegaard and Perry Laboratories, Gaithersburg, MD) at 40°C for 1 h. The bead was then washed with water. A substrate solution (0.3 ml of 0.3% ophenylene-diamine 2 HCl in 0. I M citrate-phosphate buffer (pH 5.5) containing 0.02% H202) was added (Mushahwar and Overby, 1981). The enzymic reaction was allowed to proceed for 30 min. at room temperature in the dark. The reaction was stopped by adding 1 ml of 1N H2S04. The intensity of the color that was developed as a result of the enzymic catalysis of the substrate was measured at 492 nm by using Quantum 11 Spectrophotometer (Abbott Laboratories, N. Chicago, IL). Specimens with absorbance equal to or greater than 12 times the negative control absorbance value were considered to be reactive by the criteria of this test. For this calculation, the mean absorbance value of at least 3 replicates of a seronegative specimen was used as a negative control (NC). Confirmatory tests for IgM Two confirmatory procedures were utilized to confirm the presence of antiHCV IgM. The first procedure was used to verify the presence of IgM. Reactive specimens were treated with dithiothreitol (DTT), then assayed for anti-HCV IgM. Disappearance of absorbance at 492 nm compared to the sample in a diluent without DTT signified IgM presence. The second procedure was used to verify the presence of anti-HCV core IgM. A competitive inhibition assay was performed by incubating specimens with a specimen diluent containing a synthetic core peptide (~~75) followed by the addition of a recombinant antigen coated bead. Specimens, exhibiting 50% or more reduction in absorbance at 492 nm compared to the sample without sp75 addition indicated the presence of anti-HCV core IgM.

346

Results Frequency

distribution of anti-HCV

IgM in volunteer blood donors

The frequency distribution of anti-HCV IgM in 392 healthy volunteer blood donors is shown in Fig. 1. The assay utilized as a solid-phase capture antigen polystyrene beads coated with the 3 HCV recombinant antigens (~100-3, 33c and core). Five of 392 specimens were found to be reactive for anti-HCV IgM. Only 3 (0.8%) were repeatably reactive. Further analyses showed that these 3 samples were reactive with SOD-~100-3 coated beads but unconfirmable by blocking with an E. coli derived antigen from the cl00 region (Dawson et al., 1991). Frequency donors

distribution

qf anti-HCV

IgM

in ALT-elevated

commercial

plasma

A collection of 247 plasma samples from commercial plasma donors were assayed for both IgG and IgM anti-HCV. As shown in Fig. 2, 14 (6.8%) specimens were anti-HCV IgM reactive, and all were confirmed by our confirmatory procedures. The majority of anti-HCV IgM reactive specimens analyzed so far were anti-HCV core-IgM positive.

Sample/Cutoff Fig. 1. Histogram of the distribution of sample/cutoff ratios obtained when 392 serum samples from a volunteer blood donor population were tested for anti-HCV IgM utilizing goat anti-human IgM-HRP as the probe and a polystyrene bead coated with HCV recombinant antigens (~100, 33c and core) as the solid phase. The sample/cutoff value for each specimen was calculated, and results were grouped by number of sera in each ratio range. Sample/cutoff ratios of > 1.0 are considered reactive specimens.

Sample/Cutoff Fig. 2. Histogram of the distribution of sample/cutoff ratios obtained when 247 plasma samples from an ALT-elevated commercial plasma donor population were tested for anti-HCV IgM utilizing goat antihuman IgM-HRP as the probe and a polystyrene bead coated with HCV recombinant antigens (~100, 33c and core) as the solid phase. The sample/cutoff value for each specimen was calculated, and results were grouped by number of sera in each ratio range. Sample/cutoff ratios of > 1.0 are considered reactive specimens.

Specificity studies The specificity of anti-HCV IgM assay was investigated by testing sera from volunteer blood donors (Fig. 1) and from patients with a variety of diseases other than HCV as shown in Table 1. Anti-HCV IgM was not detected in any of these sera. TABLE 1 Lack of cross-reactivity

with anti-HCV

Serum

IgM ELISA in sera positive for various antibodies No.

Anti-HCV IgG”

Acute hepatitis A Convalescent hepatitis A Acute hepatitis B Chronic hepatitis B Anti-HBc reactive Rheumatoid factorb Rubella IgM reactive EBV VCA-M reactive ANA reactive “Confirmed positive. b( IO-2000 IU/ml).

335 I4 :: 42 : I4

8 0

IgM

348 TABLE 2 Demonstration Patient

No.

of anti-HCV IgM and IgG in patients with resolved acute hepatitis C virus infection

Weeks after henatitis onset L

ALT (IUII)

Sample/cutoff

ratios (S/CO)”

IgM assays

IgG assay

Antigens ~100, Core antigen 33c, core 1

0

2 3 4 6 12 0 :

3 6 12 0

2 6 12 14 -1 1

6

-9 0 5 :: 68

Antigens ~100, 33c, core

14 17 39 274 346 1175 429

0.1 0.1 0.1 0.1 0.1 0..6 0.2

0.2 0.1 0.1 0.2 0.2 1.0 0.4

0.2 0.2 0.2 0.3 0.8 4.1 5.1

41 51 63 183 44 72

0.2 0.2 0.2 1.3 3.6 3.4

0.4 0.3 0.2 2.9 7.5 8.4

0.2 0.2 0.2 0.6 5.1 5.1

27 180 401

0.1 1.2 4.9 I.1 0.6

0.1 2.3 12.8 1.6 0.8

0.3 0.4 4.2 5.1 5.1

26 541 17 22 17 16 14 10

0.2 12.6 3.2 0.3 0.2 0.2 0.2 0.2

0.2 19.9 4.6 0.4 0.3 0.3 0.2 0.3

0.4 5.6

0.1

1.5 0.8 0.1 0.1 0.1

0.2 1.7 0.9 0.2 0.1 0.1

0.4 2.4 5.6 5.6 5.6 5.6

1.3 0.9 0.9 0.8 0.8 0.7 0.8

1.0 0.7 0.3 0.3 0.3 0.3 0.2

0.4 1.2 2.6 6.4 6.4 6.4 6.4

6:: 91 28 20 13

Ob

1 4 : 52 68 “S/CO > 1.O are considered reactive specimens. bExact date of onset is unknown.

::: 5.6 5.0 4.0 3.6

349

Acute hepatitis C infection Sequential bleedings from 6 patients with acute HCV infections followed by recovery (as determined by repeatedly normal ALT levels) were tested for both their IgM and IgG response to each of3 regions of the HCV genome (~100-3, 33c and core). The data is shown in Table 2. Five of 6 patients (patient Nos. 26) produced both IgM and IgG antibodies -against the 3 recombinant antigens. In contrast, patient no. 1 produced IgM antibodies. to only 33c (data not shown) and core antigens. In each of the 6 cases, anti-HCV IgM was detected during the early elevation of ALT. Persistence (l-2 months) varied from patient to patient and did not correlate with either ALT persistence or levels. Chronic hepatitis C infection Sequential bleeding from 29 patients categorized as having PT-NANBH was studied for both the IgM and IgG response to each of the 3 regions of the HCV genome as described above. All these patients serocokrverted for antibodies to HCV and subsequently developed a chronic HCV infection. The results of our TABLE 3 Demonstration

of anti-HCV

Patient

Weeks after hepatitis onset

7

IgM and IgG in chronic post-transfusion ALT (ukat/l)

ELISA sample/cutoff

hepatitis C patients ratios (S/CO)

IgM Assay

IgG Assays

Core antigen

Antigens (CIOO, 33c, core)

0.6 1.9 0.9 1.6

Core antigen

-7 2 z

0.17 7.40 19.80

0.2 0”:: 0.1

0.4 1.9” 3.0 1.5

I5 :;

2.07 4.37 4.65

0.1 7.8 0.2

5.6 5.6

0.6 11.4 0.5

55 70

4.03 2.46

0.4 0.3

5.6 5.6

11.4 Il.4

-10 1 3 I5 21 23 29

0.26 14.95 8.68 8.07 0.77 10.91 0.95 3.54 0.87 0.90

0.1 0.3 1.8 0.3 0.3

0.6 5.3” 5.6 5.6 5.6 5.6 5.6 5.6 5.6 5.6 5.6

0.4 Il.4 11.4 11.4 11.4 I I.4 11.4 11.4 11.4 11.4 11.4

::

1.06 ‘Passive acquisition.

00:: 0.6 0.9 0.7 0.9

350

analyses for 2 patients are summarized in Table 3. Both patients produced transient anti-HCV IgM against all 3 recombinant antigens. Among individuals who were followed for months or years after their initial hepatitis infection, anti-HCV IgM response was detected in the first weeks after the diagnosis of hepatitis. Thus, in these cases the IgM response is indicative of recent infection. In one individual (patient no. 7, Table 3), the IgM response (anti-core IgM) was not detected until 42 weeks after the onset of clinical symptoms. This patient had passively acquired anti-core in the initial weeks after transfusion which eventually subsided and was replaced by the patient’s own IgM and IgG response against core antigen first noted at week 42. Low levels of core anti-HCV IgM were detected in 5 of 66 patients (data not shown) with clinically diagnosed chronic HCV infection. It is unclear whether these specimens represent some post-IgM response as noted with patient no. 7, or are related to a reactivation phenomenon unseen previously with this disease. The extinction titer of anti-HCV IgM core was determined for all patients listed in Table 2 (acute HCV cases followed by recovery) and several of the patients who developed chronic HCV infection. The geometric mean titer of sera collected during the acute phase of the disease for those who resolved their infection was 1: 1584 while that for those who progressed to chronicity was 1:635.

Discussion The specificity of anti-HCV IgM assay was verified with sera from volunteer blood donors and sera from patients with various acute viral infections. The 3 repeatably reactive sera among the volunteer blood donors constitute a nonspecific reactivity for SOD since they were not confirmed by blocking with an E. co&derived antigen from the cl00 region (Dawson et al., 1991). Crossreactions between either IgM tests for hepatitis A, B, or EBV, or interference by rheumatoid factor and ANA were not detected signifying a high degree of specificity of this assay. Our analyses of selected commercial plasma donors with elevated ALT levels show a high prevalence (77%) of anti-HCV IgG. This is not surprising, since it is known that the prevalence of anti-HCV in unselected commercial plasma donors with normal ALT levels is greater than in volunteer blood donors (Williams and Dodd, 1990; Dawson et al., 1991). The incidence of anti-HCV IgG in Italian volunteer blood donors with high ALT levels has been reported to be 40.5% (Sircha et al., 1990). The finding that 6.8% of commercial plasma donors with high ALT levels are also anti-HCV IgM reactive, suggests that this population more often has recent or active HCV infection than the volunteer blood donor population. Since our anti-HCV IgM assay was designed to effectively eliminate interference by rheumatoid factor (Table 1) and competition with high

351

concentration of anti-HCV IgG (Table 2), this high prevalence of ongoing HCV infection in commercial plasma donors with elevated ALT level is not overestimated since all reactive specimens were confirmed by the confirmatory procedures. Our studies indicate that anti-HCV IgM is detected in sera of individuals with ongoing HCV infection and hence it is a useful serologic marker for estimating relative time of exposure to this disease. This marker was detected transiently during the acute phase of the disease in all individuals who resolved their HCV infection and also in most individuals who progressed to chronicity. The short-term persistence of anti-HCV IgM strengthens its value as a serological marker of recent acute HCV infection. In contrast, in hepatitis B virus-infected patients, anti-HBc IgM rise to high titers and persist at low titers for long periods in actively replicating chronic cases (Lemon et al., 1981; Chau et al., 1983). The exact time of appearance and disappearance and the. duration of persistence of anti-HCV IgM cannot be adequately addressed from this study since the interval between consecutive bleedings of these HCV patients varied between 2-6 weeks. In order to exactly verify these parameters, we recommend consecutive bleedings to be performed every 3 to 5 days due to the apparent transient nature of this marker. Also, it is difficult to assess the utility of this assay based on one serum sample taken from multitransfused patients that develop PT-NANBH because of the passive acquisition of both anti-HCV IgG and IgM (data not shown). Our data also indicate that the putative HCV recombinant core antigen is the best target for detecting anti-HCV IgM (Table 2). Assays performed with separate recombinant HCV antigens or synthetic peptides revealed that indeed, core antigen was the immunodominant epitope for anti-HCV IgM detection. However, further studies are needed to determine the relative importance of additional structural HCV antigens such as envelope. This study extends our knowledge of the clinical aspects of HCV infection. The development and future availability of anti-HCV IgM assay should be further explored in studies on the clinical outcome of acute HCV infections and related to other HCV markers. References Barany, G. and Merrifield, R.B. (1980) The Peptides: Analysis, Synthesis, Biology, In: E. Gross and T. Meinhoeffer (Eds) Academic Press, New York, NY, Vol. 2, pp. l-284. Chau, K.H., Hargie, M.P., Decker, R.H., Mushahwar, I.K. and Overby, L.R. (1983) Serodiagnosis of recent hepatitis B infection by IgM class anti-HBc. Hepatology 3, 142-149. Choo, Q.-L., Kuo, G., Weiner, A.J., Overby, L.R., Bradley, D.W. and Houghton, M. (1989) Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 244, 359-362. Dawson, G.J., Lesniewski, R.R., Stewart, J.L., Boardway, K.M., Gutierrez, R.A., Pendy, L., Johnson, R.G., Alcalde, X., Devare, S.G., Robey, W.G. and Peterson, D.A. (1991) Detection of antibodies to hepatitis C virus in U.S. blood donors. J. Clin. Microbial. 29, 1479-1486.

352 Desai, S., Casey, J., Braun, D., Mehta, S., Hunt, J., Dawson, G., Lesniewski, R., Peterson, D. and Devare, S. (1990) Molecular cloning an expression of HCV sequences in E. co/i and their utility in diagnostic test. Int. Sot. Blood Transfusion, Am. Ass. Blood Banks Joint Congr. Los Angeles, CA, Nov. l&15, Abstract S392. Flehmig, B., Ranke, M., Berthold, H. and Gerth, J.-J. (1979) A solid-phase radioimmunoassay for detection of IgM antibodies to hepatitis A virus. J. Infect. Dis. 140, 169-175. Kangro, H.O. (1980) Evaluation of a radioimmunoassay for IgM-class antibodies against cytomegalovirus. Br. J. Exptl. Pathol. 61, 512-520. Kuo, G., Choo, Q.-L., Alter, H.J., Gitnick, G.L., Redeker, A.G., Purcell, R.H., Miyamura. T., Dienstag, J.L., Alter, M.J., Stevens, C.E., Tegtmeier, G.E., Bonino, F., Colombo, M., Lee, W.S., Berger, K., Schuster, J.R., Overby, L.R., Bradley, D.W. and Houghton, M. (1989) An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis. Science 244, 3622364. Lemon, S.M., Gates, N.L., Simms, T.E. and Bancroft, W.H. (1981) IgM antibody to hepatitis B core antigen as a diagnostic parameter of acute infection with hepatitis B virus. J. Infect. Dis. 143, 8033809. Mattsson, L., Gutierrez, R.A., Dawson, G.J., Lesniewski, R.R., Mushahwar, I.K. and Weiland, 0. (1991) Antibodies to recombinant and synthetic peptides derived from the hepatitis C virus genome in long-term followed patients with post-transfusion hepatitis C. Scan. J. Gastroenterol. (In Press). Meurman, O.H., Viljanen, M.K. and Granfors, K. (1977) Solid-phase radioimmunoassay of rubella virus immunoglobulin M antibodies. Comparison with sucrose density gradient centrifugation test. J. Clin. Microbial. 5, 2577262. Mushahwar, I.K. and Overby, L.R. (1981) An enzyme immunoassay for hepatitis B e-antigen and antibody. J. Virol. Methods 3, 89-97. Sirchia, G., Almini, D., Bellobuono, A., Giovanetti, A.M., Marconi, M., Mercuriali, F., Mozzi, F., Parravicini, A., Pizzi, M. and Zanuso, F: (1990) Prevalence of hepatitis C virus antibodies in Italian blood donors. VOX Sang. 59, 2629. Williams, A.E. and Dodd, R.Y. (1990) The serology of hepatitis C virus in relation to posttransfusion hepatitis. Ann. Clin. Lab. Sci. 20, 1922199.