Significance of IgM Antibody to Hepatitis C Virus in Patients with Chronic Hepatitis C STEFANO BRILLANTI, CATERINAh h S C I , P I E R 0 RICCI,MARIO MIGLIOLI AND LUIGIBARBARA Department of Internal Medicine and Gastroenterology, University of Bologna, 40138 Bologna, Italy

We assessed the correlation between the positivity for serum IgM antibody to hepatitis C virus and the activity of liver disease in patients with chronic hepatitis C virus infection. Serum samples were taken from 10 antibody to hepatitis C virus-positiveasymptomatic patients with normal serum ALT levels, from 14 untreated patients with clinically and histologically proven chronic hepatitis C and from 26 patients with clinically and histologically proven chronic hepatitis C assigned to receive recombinant interferon or-2a (6 million IU three times a week for 6 mo). Each serum specimen was tested for IgM antibody to hepatitis C virus-associated C 100-3 antigen by enzyme-linked immunosorbent assay. Patients were observedfor at least 12 mo. All 10 patients with normal ALT values tested negative for IgM antibody to hepatitis C virus. In contrast, 33 of 40 (82%)patients with chronic hepatitis C had IgM antibody to hepatitis C virus, and a positive correlation was seen between the ALT level and the level of IgM antibody to hepatitis C virus (r = 0.803, p < 0.001). During interferon treatment, ALT levels declined into the normal range in 18 of 26 treatedpatients (69%) and remained norma1 after stopping treatment in 8 patients (31%). In untreated patients, in treated patients who did not respond to interferon treatment and in responder patients who relapsed, no significant changes in IgM antibody to hepatitis C virus levels were seen during the study period. In contrast, IgM antibody to hepatitis C virus became undetectable by the end of interferon treatment in seven of eight patients with a sustained response. In conclusion, we found a positive correlation between the presence of serum IgM antibody to hepatitis C virus and the activity of the hepatitis C-induced liver disease. In patients with chronic hepatitis C showing a response to or-interferon treatment, the disappearance of IgM antibody to hepatitis C virus predicted that the 1992;15: response would be sustained. (HEPATOLOGY 998-1001.)

During acute viral infections, antibody responses are characterized by an early IgM response that wanes as a

Received May 20, 1991; accepted December 27, 1991. Address reprint requests to: Dr. Stefan0 Brillanti, Clinica Medica I, Policlinico S. Orsola, Via Massarenti 9, 40138 Bologna, Italy. 31i1136633

subsequent IgG response arises. Nevertheless, IgM antibodies to viral antigens frequently persist if chronic infection evolves. Persistence of IgM antibody has been shown in both chronic type B and chronic delta hepatitis infections and in several other chronic viral diseases (1-4). Thus in patients with chronic viral hepatitis, testing for IgM antibody to specific viral antigens permits identification of patients with active viral replication and ongoing virus-induced liver disease. Commercial enzyme immunoassays for the detection of antibody to hepatitis C virus (HCV) are able to detect circulating antibodies of the IgG class only. The aims of this study were to assess an enzyme immunoassay procedure for the detection of IgM antibody to HCV (IgM anti-HCV) and to evaluate the clinical significanceof this antibody in patients with chronic HCV infection. PATIENTS AND METHODS Patients. The study group consisted of 50 adult patients (16 men and 34 women, ages 29 to 58 yr) who had serum IgG antibody to recombinant HCV-associated C100-3 antigen (anti-HCV) and who were being observed at the Department of Internal Medicine and Gastroenterology of the University of Bologna, All 50 patients were known to have had anti-HCV in serum for at least 6 mo. Patients were classified into two groups. Group 1consisted of 10 patients who had normal serum ALT and immunoglobulin levels. All were asymptomatic of liver disease. Eight of these patients had HCV RNA sequences detectable in serum. Group 2 consisted of 40 patients who had elevated serum ALT levels for at least 1yr. In all patients, liver biopsy specimens revealed histological findings compatible with CAH without cirrhosis. Infections with other hepatotropic viruses (hepatitis A, hepatitis B, Epstein-Barr virus and cytomegalovirus) were excluded by serological testing. Nonviral causes of hepatocellular injury were excluded by conventional clinical and laboratory studies. No patient had a history of blood transfusion, treatment with clotting factors, intravenous drug abuse or occupational exposure to HCV infection. These 40 patients in group 2 were defined as having chronic community-acquired or sporadic hepatitis C. Serum antibody to human immunodeficiency virus (anti-HIV) was absent in each patient. All 40 patients with chronic community-acquired hepatitis C (group 2) were observed for a period of at least 12 mo. Twenty-six were treated with 6 million IU of recombinant interferon a-2a (Roferon-A, Hoffmann-La Roche, S. A., Basel, Switzerland) given intramuscularly 3 times/wk for a 6-mo period. The 14 remaining patients were observed in a similar manner without treatment. Treated and untreated patients

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were comparable in age, sex, pretherapy ALT levels and liver histological findings. Treated patients were considered to be responders to interferon treatment if ALT levels fell into the normal range by the end of treatment (month 6). Patients were considered to be nonresponders if no significant changes were seen in the ALT levels or if some decrease occurred from the baseline but not to within the limits of the normal range by the end of treatment. Patients were observed for at least 6 mo after interferon treatment. To define the changes in levels of IgM anti-HCV during the natural course of chronic community-acquired hepatitis C both during and after interferon treatment, serial serum specimens obtained monthly were tested for this antibody and for routine laboratory biochemical determinations. Sera from a sample of 10 treated patients were also assayed for HCV RNA sequences. Serological Testing. Serum specimens were tested for anti-HCV by an enzyme immunoassay able to detect IgG antibody to the recombinant HCV antigen ((3100-3) (Ortho HCV ELISA Test Systems, Ortho Diagnostic Systems, Raritan, NJ) observing the manufacturer's instructions. All sera were retested with the complementary recombinant immunoblot assay using the cloned and expressed ClOO recombinant antigens from yeast and the 5-1-1recombinant antigens from Escherichia coli (Chiron RIBA HCV, Chiron Corp., Emeryville, CA). Sera were also retested with a qualitative enzyme immunoassay for the detection of IgG antibody to recombinant structural (C22-3)and nonstructural (C200) HCV antigens (Ortho HCV ELISA Test Systems 2nd Generation, Ortho Diagnostic Systems) observing the manufacturer's instructions. Each serum specimen was tested for IgM anti-HCV after specific removal of serum IgG using an IgG removal device with immobilized, recombinantly engineered protein G (GammaGone, Genex Corp., Gaithersburg, MD). IgM anti-HCV was then assayed by a solid-phase, antibody-capture immunoassay as follows: 200 pI of serum diluted 1:21 in PBS was added to microtiter wells coated with an HCV-derived recombinant polypeptide ((3100-31, provided by Ortho Diagnostic Systems and incubated for 2 h r at 37" C. The plates were washed with PBS, and 200 pl of antihuman IgM (p-chainspecific) (antibody isolated from goat antihuman IgM [ p-chain-specific] antiserum by immunospecific purification), conjugated to horseradish peroxidase (Sigma Chemical Co., St. Louis, MO) and diluted 1:5,000 in 3%BSA in PBS, was added. The microtiter plates were incubated for 1h r at 37" C and then washed. Finally, 200 pl of substrate solution (ophenylenediamine in citrate-phosphate buffer) was added to each well and incubated for 30 min at room temperature. The reaction was stopped by adding 50 p1 of 4 N sulfuric acid to the wells, and color intensity was measured with a microwell spectrophotometric reader at a wavelength of 492 nm and expressed as optical density values. Negative controls included specimens from healthy anti-HCV-negative patients. Positive controls included specimens from anti-HCV-positive patients with well-characterized acute posttransfusion hepatitis C. Samples with optical density (O.D.) values twice the mean value of three duplicate negative controls were considered reactive. The nature of IgM anti-HCV was confirmed in each case by the loss of reactivity after the treatment of serum samples with 2-mercaptoethanol. Finally, each IgM anti-HCV-positive serum specimen tested negative for rheumatoid factor using a latex fixation test. The enzyme immunoassay ratio of sample O.D. to negative control mean O.D. was used as a measure of IgM anti-HCV

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FIG. 1. Relationship between IgM anti-HCV (C100-3antigen) values, expressed as the enzyme immunoassay ratio of sampIe O.D. to negative control mean O.D., and ALT values in patients with chronic hepatitis C (r = 0.803, p < 0.001).

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FIG. 2. Absence of correlation between I& anti-HCV (C100-3 antigen) values, expressed as enzyme immunoassay O.D. values, and ALT values in patients with chronic hepatitis C (r = 0.068, p = NS).

level. All serum samples from 20 HBsAg-positive, IgM antiHBc-positive and anti-HCV-negative patients with chronic hepatitis B yielded enzyme immunoassay O.D. ratios of less than 1.5, whereas all serum samples from five anti-HCVpositive patients with acute posttransfusion HCV infection yielded O.D. ratios of more than 3.0. Serum HCV RNA sequences were detected using a "nested" polymerase chain reaction, as previously described (5), with nested polymerase chain reaction primers from the highly conserved 5' non-coding region. In brief, an annealing temperature of 50" C was used in the first round (35 cycles), and 46" C was used in the second round (30 cycles). RNA was prepared by the polyethylene glycol-SDS method, and complementary DNA (cDNA) was synthesized by reverse transcriptase using random primers. Statistical Analyses. Statistical analyses were performed using Student's t test and correlation coefficient analysis.

RESULTS All 50 patients included in the study (groups 1and 2) were positive for I& anti-HCV using the commercial

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FIG. 3. IgM anti-HCV (C100-3 antigen) values before, during and after interferon treatment in nonresponder patients. Values are expressed as mean 5 S.E. of the enzyme immunoassay ratios of sample O.D. to negative control mean O.D. (negative 5 2.0).

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first and second generation Ortho HCV ELISA tests. They were confirmed positive by recombinant immunoblot assay. Among the 10 asymptomatic anti-HCV-positive patients without active liver disease (group l),8 were HCV RNA positive, but none had detectable IgM anti-HCV. In contrast, 33 of the 40 (82%) patients with chronic hepatitis C (group 2) were seropositive for IgM anti-HCV. The seven patients who tested negative for IgM anti-HCV had mean ALT values that were significantly lower than those of the IgM anti-HCV-positive patients (67 2 18.4 IU/L vs. 129.2 k 14.7 IU/L, p < 0.02). In addition, in patients positive for IgM anti-HCV, a positive correlation existed between the height of the serum ALT level and the level of IgM anti-HCV (Fig. 1). In contrast, no correlation existed

FIG. 5. IgM anti-HCV (C100-3 antigen) values before, during and after interferon treatment in responder patients with relapse. Values are expressed as mean t S.E. of the enzyme immunoassay ratios of sample O.D. to negative control mean O.D. (negative 5 2.0).

between the height of the serum ALT level and the level of IgG anti-HCV (Fig. 2). During the study period, ALT values remained elevated in all 14 untreated patients. In contrast, ALT levels fell into the normal range in 18 of 26 patients (69%) treated with a-interferon, and remained normal after stopping treatment in 8 patients (31%). All but 1of the 18 patients (94%) who had a response to a-interferon treatment tested positive for IgM antiHCV at the start of the study, whereas IgM anti-HCV was present in only 5 of 8 (62%)nonresponder patients. Ten of the 14 untreated patients tested positive for IgM anti-HCV. These patients had no significant changes in IgM anti-HCV levels during the study period. Similarly, in the five IgM anti-HCV-positive treated patients who did not respond t o a-interferon treatment, no significant changes were seen in IgM anti-HCV levels by the end of treatment (Fig. 3). A different pattern of IgM anti-HCV O.D. ratios was observed among the eight patients who responded to interferon treatment and maintained normal ALT levels after withdrawal of therapy. Initially, all eight patients were positive for IgM anti-HCV. With treatment, serum levels fell in all, becoming undetectable in seven patients (Fig. 4). Detection of HCV RNA in serum from four of these patients revealed the HCV RNA sequences in serum before treatment and the disappearance of the HCV genome in three patients by the end of the therapy. In contrast, serum IgM anti-HCV levels remained elevated in all patients who responded transiently but who had a relapse after stopping interferon treatment. In some patients, a slight reduction was seen in IgM anti-HCV levels during treatment, but levels of this antibody generally returned to the pretreatment values afterward (Fig. 5 ) . Six of these patients were tested for serum HCV RNA sequences; viral genome was present before interferon treatment and became undetectable in four patients by the end of interferon therapy.

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DISCUSSION The results of this study indicate that most patients with chronic hepatitis C have circulating IgM antibody to the HCV-associated nonstructural C100-3 antigen. This IgM antibody was found in 82% of anti-HCVpositive patients with clinically and histologically proven chronic hepatitis. In contrast, all patients with circulating IgG anti-HCV and HCV RNA sequences but without evidence of active liver disease tested negative for IgM anti-HCV. Furthermore, we previously described that 10 prospectively observed transfusion recipients with acute hepatitis C tested positive for IgM anti-HCV (6).These findings suggest that the presence of IgM anti-C100-3 identifies those patients with active HCV infection and HCV-induced liver disease. The mere presence of HCV infection without HCV-induced liver damage does not seem to induce a continued production! of specific IgM antibody to the HCV-associated, nonstructural C100-3 protein. Levels of IgM anti-HCV, as assessed by O.D. ratios, correlated with biochemical evidence of active liver disease. Levels of IgM anti-HCV were highest in patients with high levels of ALT and were lowest or undetectable in patients with low or normal ALT values. Interestingly, we recently demonstrated that HCV RNA levels did not correlate with ALT levels in patients with chronic community-acquired hepatitis C (7). These results support the role of the active host’s immune response to HCV replication in the pathogenesis of the liver injury in hepatitis C. Testing of sequential serum specimens indicated that spontaneous remissions in chronic hepatitis C were rare. During the study period, ALT values remained consistently higher than the upper limit of the normal range in all untreated patients with chronic hepatitis C. In addition, no significant changes were seen in IgM anti-HCV levels over the 12-mo study period. Similarly, IgM anti-HCV levels did not change significantly in patients who did not respond to a-interferon treatment. These findings suggest that the presence of IgM anti-HCV characterizes sustained active HCV infection with associated chronic virus-induced hepatitis. It is important to note that, by the end of a-interferon treatment, IgM anti-HCV fell t o undetectable levels in seven of the eight patients who had a sustained response t o a-interferon treatment. In patients who had a relapse after a response to treatment, IgM anti-HCV did not fall to undetectable levels, despite the temporary fall in ALT levels. These results suggest that, during interferon treatment of chronic hepatitis C, the disappearance of IgM anti-HCV predicts a complete remission of disease

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activity and a sustained remission after discontinuation of treatment. These data also confirm results from a previous study (8)indicating that HCV RNA,detected by polymerase chain reaction, disappears from serum after effective treatment with a-interferon, but the disappearance of HCV RNA does not seem to predict long-term response to therapy. In conclusion, IgM antibody to HCV-associated C100-3 antigen can be used as a serological marker to indicate the presence of active HCV-induced hepatitis. A sensitive test for IgM anti-HCV is needed to distinguish between asymptomatic patients who have an underlying unrecognized HCV-induced liver disease and those without accompanying liver disease. Because liver disease in the anti-HCV-positive patient may be unrelated to HCV but caused by a superimposed and unrelated form of liver injury, testing for IgM anti-HCV may provide the specific diagnosis of type C hepatitis by identifying anti-HCV-positive patients with HCV infection and an active immune response to the virus. Testing of sequential serum specimens for IgM anti-HCV in patients treated with a-interferon may also predict which patients will have a long-term response and when interferon can be withdrawn without a subsequent relapse in disease. REFERENCES 1. Banninger P, Altorfer J , Frosner GG, Pirovino M, Gudat F, Bianchi L, Grob PJ, et al. Prevalence and significance of anti-HBc IgM (radioimmunoassay) in acute and chronic hepatitis B and in blood 1983;3:337-342. donors. HEPATOLOGY 2. Sjogren M, Hoofnagle JH. Immunoglobulin M antibody to hepatitis B core antigen in patients with chronic type B hepatitis. Gastroenterology 1985;89:252-258. 3. Brunetto MR. Arrieoni A. Toti M. Almi P. Zanetti A. Ferroni P. Doris R, et al. Th;e diagnostic significance of IgM antibody to hepatitis B core antigen, revisited. Ital J Gastroenterology 1988; 20:167-170. 4. Govindarajan S, Gupta S, Valinluck B, Redeker G . Correlation of IgM anti-hepatitis D virus (HDV) to HDV RNA in sera of chronic HDV. HEPATOLOGY 1989;10:34-35. 5. Garson JA, Tuke PW, Makris M, Briggs M, Machin SJ,Preston FE, Tedder RS. Demonstration of viraemia patterns in haemophiliacs treated with hepatitis C virus contaminated factor VIIJ concentrates. Lancet 1990;336:1022-1025. 6. Brillanti S, Miglioli M, Di Febo G, Barbara L. Serum IgM antibody to hepatitis C virus in subjects with acute and chronic non-A, non-B hepatitis [Abstract]. Ital J Gastroenterol 1990;22:165. 7. Brillanti S, Garson JA, Tuke PW, Ring C, Briggs M, Masci C, Miglioli M, et al. The effect of alpha interferon therapy on hepatitis C viraemia in community acquired chronic non-A, non-B hepatitis: a quantitative PCR study. J Med Virol 1991;34:136-141. 8. Shindo M, Di Bisceglie AM, Cheung L, Shih JW,Cristiano K, Feinstone SM, Hoofnagle JH. Decrease in serum hepatitis C viral RNA during alpha-interferon therapy for chronic hepatitis C. Ann Intern Med 1991;115:700-704.

Significance of IgM antibody to hepatitis C virus in patients with chronic hepatitis C.

We assessed the correlation between the positivity for serum IgM antibody to hepatitis C virus and the activity of liver disease in patients with chro...
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