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Immunoglobulin M antibody to hepatitis B core antigen in patients with chronic type B hepatitis
GASTROENTEROLOGY
1985;89:252-8
Immunoglobulin M Antibody to Hepatitis B Core Antigen in Patients With Chronic Type B Hepatitis MARIA
SJOGREN and JAY H. HOOFNAGLE
Department of Virus Diseases, Walter Reed Army Institute of Research, Washington, DC. and the Liver Diseases Section, Digestive Diseases Branch, National Institute of Arthritis, Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland
Serum samples from 130 persons who were seropositive for hepatitis B surface antigen and who had various forms of accompanying liver disease were tested for immunoglobulin M (IgM) antibody to hepatitis B core antigen. In 99% of patients with hepatitis B e antigen-positive chronic type B hepatitis, IgM antibody to hepatitis B core antigen was present. This antibody was not present in “healthy” hepatitis B surf&e antigen carriers and was detectable in only 30% of patients with 6 hepatitis. Testing of serial sera from 38 patients with chronic type B hepatitis revealed that IgM antibody to hepatitis B core antigen persisted in patients who had evidence ofpersistent hepatitis B virus replication but ultimately disappeared in those patients who exhibited a sustained loss of serum markers of viral replication [hepatitis B virus deoxyribonucleic acid and deoxyribonucleic acid polymerase activityl. These findings suggest that the presence of IgM antibody to hepatitis B core antigen in chronic hepatitis B surface antigen carriers indicates an active immune response to persistent viral replication. Antibody responses during acute viral infections are characterized by an early immunoglobulin M (IgM) antibody response that gradually wanes as a subsequent immunoglobulin G (IgG) response arises. This pattern of antibody class responses has been shown
Received October 5, 1984. Accepted February 11, 1985. Address requests for reprints to: Jay H. Hoofnagle, M.D., Liver Diseases Section, Building 10, Room 4-D-52, National Institutes of Health, Bethesda, Maryladd 20205. The opinions and assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense. The authors thank Jeanne G. Waggoner and Lee Sosebee for technical assistance. 0 1965 by the American Gastroenterological Association 0016-5085/85/$3.30
to occur in almost all acute viral infections including the various forms of viral hepatitis-type A, type B, and 6 hepatitis (l-7). Testing for IgM antibody to specific viral antigens has been proposed as a serologic means of diagnosis of the type of viral hepatitis (Z-6).Less well characterized are the antibody class responses that occur during chronic viral infections. If viral replication becomes chronic, IgM antibodies to viral antigens frequently persist. The persistence of IgM antibody has been shown in both chronic type B and chronic &hepatitis infections (6-10)as well as in several other chronic viral diseases such as congenital rubella (ll),subacute sclerosing panencephalitis (l2), and chronic infectious mononucleosis (131.
While investigating the IgM antibody response to hepatitis B core antigen (IgM anti-HBc) among chronic hepatitis B surface antigen (HBsAg) carriers, we have found that this antibody response defines several features of the natural history of chronic type B hepatitis. Patients with persistence of viral replication and serum biochemical evidence of ongoing chronic hepatitis maintained detectable serum levels of IgM anti-HBc. Patients who underwent a remission in disease activity and who lost detectable markers of viral replication remained HBsAg-positive, but usually lost IgM anti-HBc. The presence of detectable IgM anti-HBc, therefore, was a sensitive marker of active viral replication and indicated that the accompanying liver disease was due to the hepatitis B virus (HBV) infection. The absence of this antibody indicated a lack of HBV replication and suggested that any accompanying hepatitis was due to a superimposed, unrelated or secondary form of
Abbreviations used in this paper: IgM, IgG, immunoglobulins M and G, respectively; IgM anti-HBc, immunoglobulin M antibody to hepatitis B core antigen; HBV-DNA, hepatitis B virusdeoxyribonucleic acid.
August 1985
IgM ANTI-HBc
liver disease (type A, non-A, non-B, or 6 hepatitis drug-induced liver injury].
or
Materials and Methods Patient Population The study comprised 130 patients who had HBsAg in serum and who were being followed at the National Institutes of Health Clinical Center. All 130 patients were known to have had HBsAg in serum for at least 6 mo before this serologic and clinical evaluation. Patients were classified into four groups. Group 1 consisted of 100 patients who were seropositive for the hepatitis B e antigen (HBeAg) and who had elevations in the serum alanine aminotransferase levels (ALT) [mean ALT 253, range 4% 1370 U/L]. Superinfection with the HBV-associated 6 agent was excluded in these patients by the absence of anti-3 in the serum. These 100 patients in group 1 were defined as having HBeAg-positive chronic type B hepatitis. Group 2 consisted of 10 patients who were seropositive for antibody to the hepatitis B e antigen (anti-HBe) and who had normal serum ALT levels (mean ALT 29, range 15.-36 U/L). These patients were defined as asymptomatic “healthy” HBsAg carriers without evidence of active liver disease. Group 3 consisted of 10 patients who were seropositive for anti-HBe but who, nevertheless, had elevated serum ALT levels (mean ALT 163, range 48-414 U/L). In 9 of these patients, a liver biopsy specimen was taken; all specimens revealed histologic evidence of chronic hepatitis. These patients were considered to have anti-HBe-positive chronic type B hepatitis. Group 4 consisted of 10 patients who were seropositive for antibody to the 6 agent (in titers > 1: 100) and who had elevated serum ALT levels [mean ALT 293, range 63-1065 U/L). These patients were defined as having chronic 6 hepatitis (7). To define the behavior of IgM anti-HBc during the course of chronic type B hepatitis, serial serum specimens from 38 of the patients from group 1 (HBeAg-positive) were tested for this antibody. These 38 patients have been followed for l-9 yr [mean 3.1 yr). The patients were routinely seen every l-6 mo and had blood tested for serologic markers of HBV infection as well as routine laboratory biochemical tests including ALT, aspartate aminotransferase, bilirubin, albumin, and globulin using automated techniques (SMAC: Technicon Instruments Corp. Tarrytown, N.Y.). Serologic
Testing
Serum specimens were tested for HBsAg and antibody (anti-HBs) by radioimmunoassays (Ausria-II and Ausab: Abbott Laboratories, North Chicago, Ill.). Serum HBeAg and anti-HBe were assayed by immunodiffusion and radioimmunoassay (HBeAg Test, Abbott) (14,15). Serum hepatitis B virus-deoxyribonucleic acid (HBV-DNA) was measured by molecular hybridization using a 32Plabeled HBV-DNA probe [Bethesda Research Labs, Bethesda, Md.) (16). Hepatitis B virus-DNA polymerase activity was assayed by determining [3H]thymidine incorporation (17). A single specimen from each patient was also tested for antibody to the 6 agent (anti-61 by solid-phase radio-
253
immunoassay (courtesy of Dr. John Gerin and Ronald Engle) (7). Immunoglobulin M antibody to hepatitis B core antigen was assayed by a solid-phase, antibody-capture radioimmunoassay technique (3). Polyvinyl microtiter plates were coated with goat antihuman IgM (w-chain specific]. Fifty microliters of serum diluted 1:100 in phosphatebuffered saline was added to the microtiter wells and incubated for 4 h at 30°C. The plates were washed with phosphate-buffered saline and 25 ~1 of a preparation of hepatitis B core antigen purified from chimpanzee liver was added. The microtiter plates were incubated overnight and then washed. Finally, 40 ~1 of ‘251-labeled human IgG anti-HBc (200,000 cpm) was added to each well and left for 4 h at 4°C. The plates were washed again, the wells cut out, and the radioactivity adhering to the microtiter wells was counted in a y-counter. Controls included specimens from normal individuals without HBV infection. Samples were also tested without the addition of purified hepatitis B core antigen so as to detect rheumatoid-factor-like activity that might yield false-positive results. The radioimmunoassay ratio of sample counts per minute to negative control mean counts per minute was used as a measure of IgM anti-HBc level. Negative samples yielded radioimmunoassay ratios of ~2.1, whereas strongly positive samples, such as those from patients with acute type B hepatitis, yielded ratios of >15 (3). Single samples from each patient were also tested for IgM anti-HBc using a commercial enzyme immunosorbent assay (Corzyme-M, Abbott) in which samples are assayed at a dilution of -1:lOOO (4). Percutaneous liver biopsies had been performed on 93 of these 130 patients, including 78 from group 1, none from group 2, 9 from group 3, and 6 from group 4. The biopsy specimens were categorized by standard histologic criteria as either chronic persistent hepatitis, chronic active hepatitis, or cirrhosis (18).
Statistical
Analyses
Group frequencies were compared using the x2 test. Values within groups were compared by means of correlation coefficient analysis. Changes in laboratory test results in individual patients were analyzed by the paired Student’s t-test.
Results Results of testing sera for IgM anti-HBc in the four groups of patients are shown in Table 1. All except 1 patient with HBeAg-positive chronic type B hepatitis were seropositive ,for IgM anti-HBc by radioimmunoassay. The single exception was an asymptomatic Asian man who had been known to be HBsAg-positive for many years and whose serum ALT levels were persistently less than twice the upper limit of the normal range. Of the 100 HBeAgpositive patients, 99 had detectable HBV-DNA in serum and 97 had DNA polymerase activity.
GASTROENTEROLOGY
SJOGREN AND HOOFNAGLE
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Table
1. Results of Testing Four Groups of Hepatitis B Surface Antigen-Positive Patients for lmmunoglobulin M Antibody to Hepatitis B Core Antigen by Radioimmunoassay No. nositive
or test results for IgM antiHBc
Group HBeAg+ chronic type B Healthy HBsAg+ carriers Anti-HBe+ chronic type B 6 Hepatitis
No. tested
HBeAg+
HBV-DNA+ or DNAp+
No. +
Mean level”
100
100
99
99
6.2
10
0
0
1
1.7
10
0
5b
10
9.0
10
3
3
3
2.7
HBsAg, hepatitis B surface antigen; anti-HBc, antibody to hepatitis B core antigen; HBeAg, hepatitis B e antigen; anti-HBe, antibody to HBeAg; HBV-DNA, hepatitis B virus-deoxyribonucleic acid; DNAp, DNA polymerase activity. ” Level expressed as radioimmunoassay ratio units = sample counts per minute/ negative control mean counts per minute: >2.1 is considered positive. b Intermittently positive.
The HBsAg-positive patients who were negative for fiBeAg were less likely to have detectable IgM anti-HBc. Among 10 patients who were considered to be healthy HBsAg carriers without active liver disease (group 2), only 1 was weakly seropositive for this IgM antibody. None had detectable HBV-DNA or DNA polymerase in serum. In contrast, the 10 patients with anti-HBe who had elevated ALT levels (group 3) were all positive for IgM anti-HBc, often at high levels. Five of these patients were intermittently positive for HBV-DNA and DNA polymerase at low levels. The 10 patients with 6 hepatitis (group 4) were Table
2. Correlations Between Immunoglobulin Evidence of Chronic Hepatitis Disease
IgM anti-HBc radioimmunoassay ratio results”
No.
Negative (<2.1)
10
usually negative for IgM anti-HBc. Three patients had low levels of IgM anti-HBc. Although sera from a total of 113 of the 130 patients were reactive for IgM anti-HBc using radioimmunoassay, sera from only 2 were reactive for this IgM antibody using the commercial enzyme-linked immunosorbent assay. These 2 patients were in the midst of a severe exacerbation of their disease at the time. Both patients had serum ALT levels >800 u/L and 1 patient was jaundiced at the time that they were seroreactive for this antibody. Both patients eventually improved and this reactivity disappeared. The level of IgM anti-HBc as detected by radioimmunoassay correlated to spme extent with the severity and the activity of the underlying chronic type B hepatitis. Thus, the mean of radioimmunoassay ratio units in patients with cirrhosis was higher (mean f SD = 9.7 ‘_ 4.6) than in those patients with chronic persistent hepatitis (6.9 t 2.8; p < 0.05) or chronic active hepatitis (7.3 ? 3.1; p < 0.05). In addition, there was a positive correlation between the height of serum ALT and aspartate aminotransferase levels and the level of IgM aptiHBc (Table 2). This correlation between IgM antiHBc and serum biochemical evidence of activity of liver disease was most marked among HBsAg-positive patients with anti-HBe. Furthermore, this correlation did not hold true for patients with S hepatitis; many of the patients with 6 hepatitis had cirrhosis (70%) and marked elevations in serum ALT and aspartate aminotransferase levels, yet these 10 patients were either seronegative for IgM anti-HBc or had low levels of the antibody. Finally, although there was a correlation between the presence of IgM anti-HBc and the presence of HBV-DNA and DNA
M Antibody to Hepatitis Activity in 120 Hepatitis
B Core Antigen Levels and Biochemical B Surface Antigen-Positive Patients
Mean (SD] of serum ALT (U/L]
AST (U/L)
Albumin (gldl)
Protime (s)
4.5
11.6
(1;) 117
(0.2) 4.3
CO.51 12.1
2.1-4.9
25
(ii, 239
5.0-9.9
63
(2411 218
(167) 111
(0.31 4.0
(0.9) 13.3
10.0-14.9
17
(1961 275
(136) 137
(0.4) 4.0
(1.2) 12.9
5
(2271 498
(115) 250
(0.4) 3.8
(1.51 13.6
(0.41
Cl.41
>15.0
(3611
Correlation coefficient p Value
Vol. 89, No. 2
(151)
0.261
0.226
-0.387
co.02
0.325
DNAP (cpm) 129 (261) 2796 (2714) 2367 (2881) 1655 (1392) 2253 (2611) 0.014
(r)b <0.005
NS
Anti-HBc, antibody to hepatitis B core antigen; ALT, alanine aminqtransferase; AST, aspartate aminotransferase: protime, prothrombin time; DNAp, DNA polymerase; cpm, counts per minute; NS, not significant or p > 0.05. a Radioimmunoassay ratio = sample counts per ratio minute/negative control counts per minute: >2.1 is positive. b Correlation coefficient between the IgM anti-HBc radioimmunoassay units and the biochemical laboratory result analyzed.
August
1985
IgM ANTI-HBc
Table 3. Serologic Markers in
38
255
Patients With Chronic Type B Hepatitis at the Start and End of Follow-up
Evaluation IgM Group Persistent
(No.]
Time
HBeAg
ALT” (U/L)
DNAp”
HBeAg
Start End Start End Start End
+ + + _
125 (239) 183(?164) 446 (2313) 35 (211)" 150 (267) 123 (263)
3558(+1860) 4459(?3673) 3573(?4037) 28 (211)" 2014 (22833) 1578(t3390)
(20) Seroconversion (13) Reactivation (51
+ +
(cpm)
anti-HBc” (RIA ratio) 7.9 (k3.0) 8.2 (k3.5) 10.3 (24.5) 1.9 (21.3)" 9.4 (25.2) 7.9 (22.8)
HBeAg, hepatitis B e antigen; ALT, alanine aminotransferase activity (normal ~45 U/L); DNAp, DNA polymerase (negative
polymerase in serum, there was no correlation between the level of IgM anti-HBc and the level of the HBV markers in serum. Serial specimens from 38 patients with chronic HBeAg-positive hepatitis were tested for IgM antiHBc by radioimmunoassay. These patients were selected to represent three different outcomes of HBeAg-positive chronic type B hepatitis (14). Twenty patients remained HBeAg-positive and continued to have elevated serum ALT levels (persistent HBeAg). In all 20 of these patients, IgM anti-HBc remained detectable in the serum and titers did not change appreciably (Table 3 and Figure 1).Thirteen of the 38 patients became HBeAg-negative and seroconverted to anti-HBe during the period of follow-up evaluation (HBeAg seroconversion). Associated with
this seroconversion, serum HBV-DNA and DNA polymerase activity became undetectable and serum ALT levels decreased. The serum levels of IgM antiHBc fell in all 13 patients and became undetectable in 12 (Figure 2). In these patients, the fall in serum IgM anti-HBc into the negative range occurred 0.5-5 yr (mean 1.9 yr) after the disappearance of HBeAg. The remaining 5 patients became HBeAg-negative, but serum ALT levels improved only temporarily. These 5 patients exhibited a return of biochemical evidence of disease concurrent with a return of HBVDNA and DNA polymerase in serum (spontaneous reactivation of chronic type B hepatitis) (19).Serum IgM anti-HBc reactivity remained present in all 5 patients and levels of this antibody usually increased during the period of exacerbation (Figure 3).
3 3 a
200
ALT
150
DNA Polymerase
1979
1980
1981
1982
1983
YEAR Figure
1
Serum biochemical and serologic course of a patient with chronic type B hepatitis who remained hepatitis B e antigen (HBeAg)-positive for 5 yr. Serum alanine aminotransferase levels (ALT) were persistently elevated and DNA polymerase activity and immunoglobulin M antibody to hepatitis B core antigen (IgM anti-HBc) remained present. cpm, counts per minute; RIA ratio, radioimmunoassay ratio.
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SJOGREN
GASTROENTEROLOGY
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1980
1991
1992
Vol. 89, No. 2
1983
YEAR Figure
2. Serum biochemical and serologic course of a patient with chronic type B hepatitis who became seronegative for hepatitis B e antigen (HBeAg) and developed antibody to HBeAg (anti-HBe) during the period of observation. Serum DNA polymerase activity became negative immediately before HBeAg became undetectable and, shortly thereafter, serum alanine aminotransferase (ALT) levels fell into the normal range. Serum immunoglobulin M antibody to hepatitis B core antigen (IgM anti-HBc) levels gradually decreased, becoming undetectable 18 mo after HBeAg became negative.
T
nIgM
Anti-HBc
DNA / Polymerase
I _J lo
Boo 400
5
200 2
100 1980
1981
1982
1983
YEAR Figure
3. Serum biochemical and serologic course of a patient with chronic type B hepatitis who became seronegative for hepatitis B e antigen (HBeAg) and developed antibody to hepatitis B e antigen (anti-HBe) and concurrently had an improvement in serum aminotransferase (ALT) levels. With the loss of HBeAg, serum levels of immunoglobulin M antibody to hepatitis B core antigen (IgM anti-HBc) began to fall. Subsequently, however, this patient had a reactivation of disease with return of biochemical evidence of chronic hepatitis and serologic evidence of viral replication (DNA polymerase activity and HBeAg). This spontaneous exacerbation of disease was accompanied by a marked rise in IgM anti-HBc.
August
1985
Discussion The results of this study indicate that the majority of patients with chronic type B hepatitis continue to produce detectable amounts of IgM antibody to the core component of the HBV. This antibody was found in 99% of patients who had HBeAg detectable in serum and in all of a small number of patients who were seronegative for HBeAg but who, nevertheless, appeared to have continued chronic type B hepatitis. These findings suggest that the continued presence of hepatitis B core antigen induces a continued production of specific IgM antibody. Previous reports have proposed that IgM anti-HBc could be used as a serologic marker for acute type B hepatitis (3-6).During acute infection, levels of IgM anti-HBc are usually quite high, whereas levels of IgG anti-HBc are low. With resolution of the disease, levels of IgM anti-HBc begin to fall, and this antibody is usually undetectable 3-24 mo later. In the present study, IgM anti-HBc was shown to persist in patients with chronic HBV infection, a finding that would seem to obviate the use of this antibody as a reliable marker for acute infection. However, the radioimmunoassay used in the current study differed in several important details from those immunoassays for IgM anti-HBc that are used to make the diagnosis of acute type B hepatitis. Thus, the commercial enzyme-linked immunosorbent assay was designed to only detect high titers of IgM anti-HBc such as occur during acute type B hepatitis. The assay uses a high dilution of test sample (-1: 1000) and concentrations of reagent antigen and antibody that facilitate detection of high and not low levels of IgM anti-HBc. In contrast, the radioimmunoassay used in the current study was designed to be a sensitive assay for IgM anti-HBc using the concentrations of anti-IgM and hepatitis B core antigen and the dilution of serum (1:100) that yield maximal sensitivity and specificity (3). For these reasons, these two assays are not easily comparable. Furthermore, the commercial immunosorbent assay cannot be readily adapted for the detection of IgM anti-HBc that occurs in chronic type B hepatitis. The finding of moderate or low titers of IgM antiHBc in patients with chronic type B hepatitis has been previously reported by several groups of investigators using a variety of immunologic assays for this antibody (5,6,8-10). This IgM antibody has been shown to differ from that found during acute type B hepatitis in that it is often 7-8s IgM rather than the typical 1% antibody (20,21). The current study differed from previous studies in that the IgM antiHBc was assayed not just in single serum samples but also in serial serum specimens from patients with chronic type B hepatitis. This allowed for
IgM ANTI-HBc
257
analysis of the correlation between changes in IgM anti-HBc levels and changes in biochemical and serologic features of the disease. This testing of sequential samples demonstrated that this antibody persisted in the serum of patients with persistent viral replication (continued presence of HBeAg, HBV-DNA, and DNA polymerase); but that it disappeared from the serum of those persons who lost serologic evidence of active viral replication even though these patients remained positive for HBsAg. Thus, persistence of IgM anti-HBc in the serum appeared to correlate with serologic evidence of continued viral replication. Several serologic tests have been proposed as sensitive markers of active HBV replication. Chief among these has been HBeAg, the presence of which correlates well with high levels of HBV in serum and high degrees of infectivity (14).However, HBeAg is not present in the serum of all patients who continue to produce HBV particles. Other investigators have suggested that HBV-DNA or DNA polymerase may be more direct and perhaps more sensitive markers of the production of HBV (16,17,19,22,23). Indeed, using these two serologic tests one can sometimes detect HBV in serum that contains anti-HBe and no detectable HBeAg. As shown here and by other investigators, however, even these direct markers of HBV production are not invariably present when there is serum biochemical and hepatic histologic evidence of continued chronic type B hepatitis activity (23-25).In this situation, however, IgM anti-HBc is usually detectable. In the current study, IgM antiHBc was found in all patients in a group of 10 who did not have HBeAg in serum but who did have clinical, serum biochemical, and histologic evidence of chronic hepatitis. In this same group, HBV-DNA and DNA polymerase activity were present in the serum of some patients, but only intermittently and at low titer. The presence of IgM anti-HBc, therefore, appeared to mirror the presence of even low levels of HBV production. Interestingly and importantly, however, the level of IgM anti-HBc did not correlate with the titer of HBV produced (as quantified by the level of HBV-DNA or DNA polymerase in serum). Instead, the level of IgM anti-HBc correlated better with histologic and biochemical evidence of active liver disease (10). Levels of IgM anti-HBc were highest in patients with active liver disease and were absent or low in patients who were healthy carriers or who had mild forms of chronic type B hepatitis. Testing of sequential serum specimens indicated that remissions in the chronic liver disease were accompanied by decreases in level of IgM anti-HBc. Typically, IgM anti-HBc fell to undetectable levels between 12 and 24 mo after the chronic hepatitis resolved. This
258
SJOGREN
AND
HOOFNAGLE
delay in disappearance of IgM anti-HBc after improvement in chronic type B hepatitis is another reason why testing of single serum specimens may yield conflicting results. The delay in fall of specific IgM anti-HBc after disappearance of active viral replication merely reflects the delay in disappearance of antibody responses after antigen challenge. From these data, one could hypothesize that the presence of IgM anti-HBc reflects both the presence of continued viral replication as well as an immune response to that replication. Accordingly, patients who produce high titers of virus but who have little in the way of an immune response to the virus would tend to have low or mediocre titers of IgM anti-HBc. In contrast, patients who have an active, accompanying immune response to the viral replication would tend to have high levels of IgM anti-HBc. This interpretation also would afford an explanation for the high titers of IgM anti-HBc that are found during acute viral hepatitis in which the immune response to the virus is typically strong and usually leads to complete elimination of the viral infection. Thus, IgM anti-HBc can be used as a serologic marker to indicate the presence of continued HBV replication as well as an immune response to the virus. The detection of this antibody is especially helpful in patients who have no detectable HBeAg in serum but who do have active accompanying liver disease. If the liver disease is due to HBV, IgM antiHBc should be present. If the liver disease in the HBsAg-positive and anti-HBe-positive patient is not due to HBV but is due to a superimposed and unrelated form of liver disease (such as S hepatitis or perhaps drug-induced liver injury], IgM anti-HBc should be absent.
References 1. Locarnini SA, Ferris AA, Lehmann NI, Gust ID. The antibody response following hepatitis A infection. Intervirology 1977; 8:309-18. 2. Decker RH, Kosakowski SM, Vanderbilt AS, Ling CM, Chairez R, Overby LR. Diagnosis of acute hepatitis A by HAVAB-M, a direct radioimmunoassay for IgM anti-HAV. Am J Clin Path01 1981;76:140-7. 3. Lemon SM, Gates NL, Simms TE, Bancroft WH. IgM antibody to hepatitis B core antigen as a diagnostic parameter of acute infection with hepatitis B virus. J Infect Dis 1981;143:803-9. 4. Chau KH, Hargie MP, Decker RH, Mushahwar IK, Overby LR. Serodiagnosis of recent hepatitis B infection by IgM class anti-HBc. Hepatology 1983;3:142-9. 5. Kryger P, Mathiesen LR, Aldershvile J, Nielsen JO, The Copenhagen Hepatitis Acuta Programme. Presence and meaning of anti-HBc IgM as determined by ELISA in patients with acute type B hepatitis and healthy HBsAg carriers. Hepatology 1981;1:233-7. 6. Shimizu M, Ohyama M, Takahaski Y, et al. Immunoglobulin M antibody against hepatitis B core antigen for the diagnosis
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of fulminant type B hepatitis. Gastroenterology 1983;84:60410. 7. Smedile A, Lavarini C, Crivelli 0, et al. Radioimmunoassay detection of IgM antibodies to the HBV-associated delta (8) antigen. Clinical significance of 8 infection. J Med Virol 1982;9:131-8. 8. Lemon SM, Hoofnagle JH. IgM antibody to hepatitis B core antigen in patients with chronic type B hepatitis. In: Szmuness W, Alter HJ, Maynard JE, eds. Viral hepatitis. 1981 International Symposium. Philadelphia: Franklin Institute Press, 1981:723-4. 9. Hoofnagle JH. Specific IgM and IgG antibody responses during type B hepatitis. In: Berk PD, Chalmers TC, eds. Frontiers in liver disease. New York: Thieme-Stratton, 1981:208-12. 10. Banninger P, Altorfer J, Froesner GG, et al. Prevalence and significance of anti-HBc IgM (radioimmunoassay) in acute and chronic hepatitis B and in blood donors. Hepatology 1983;3:337-42. 11. Alford CA. Studies on antibody in congenital rubella infections. Am J Dis Child 1965;110:455-63. 12. Kiessling WR, Hall WW, Yung LL, Ter Meuten V. Measlesvirus specific immunoglobulin M response in subacute sclerosing panencephalitis. Lancet 1977;i:324-7. 13. Tobi M, Morag A, Ravid Z, et al. Prolonged atypical illness associated with serological evidence of persistent EpsteinBarr virus infection. Lancet 1982;i:61-4. 14. Hoofnagle JH, Dusheiko GM, Seeff LB, Bales ZB, Waggoner JG, Jones EA. Seroconversion from hepatitis B e antigen to antibody in chronic type B hepatitis. Ann Intern Med 1981; 94:744-8. 15. Krugman S, Overby LR, Mushahwar IL, Ling CM, Froesner GG, Dienhardt F. Viral hepatitis, type B. Studies on natural history and prevention re-examined. N Engl J Med 1979; 300:101-7. 16. Berninger M, Hammer M, Hoyer B, Gerin JL. An assay for the detection of the DNA genome of hepatitis B virus in serum. J Med Virol 1982;9:57-68. 17. Kaplan PM, Greenman RL, Gerin JL, Purcell RH, Robinson WS. DNA polymerase associated with human hepatitis B antigen. J Virol 1973;12:995-1005. 18. International Group. Acute and chronic hepatitis revisited. Lancet 1977;ii:914-9. 19. Davis GL, Hoofnagle JH, Waggoner JG. Spontaneous reactivation of chronic type B hepatitis. Gastroenterology 1984;86: 230-5. 20. Sjogren M, Lemon SM. Low molecular weight IgM antibody to hepatitis B core antigen in chronic hepatitis B virus infections. J Infect Dis 1983;148:445-51. 21. Tsuda F, Naito S, Takai E, et al. Low molecular weight (7s) immunoglobulin M antibody against hepatitis B core antigen in the serum for differentiating acute from persistent hepatitis B virus infection. Gastroenterology 1984;87:159-64. 22. Bonino F, Hoyer B, Nelson J, Engle R, Verme G, Gerin JL. Hepatitis B virus DNA in the sera of HBsAg carriers. A marker of active hepatitis B virus replication in the liver. Hepatology 1981;1:386-91. 23. Hadziyannis SJ, Lieberman HM, Karvountzis GG, Shafritz DA. Analysis of liver disease, nuclear HBcAg, viral replication, and hepatitis B virus DNA in liver and serum of HBeAg vs. anti-HBe positive carriers of hepatitis B virus. Hepatology 1983;3:656-62. 24. Alberti A, Tremolada F, Fattovich G, Bortolotti F, Realdi G. Virus replication and liver disease in chronic hepatitis B virus infection. Dig Dig Sci 1983;28:962-6. 25. Kam W, Rall LB, Smuckler EA, Schmid R, Rutter WJ. Hepatitis B viral DNA in liver and serum of asymptomatic carriers. Proc Nat1 Acad Sci USA 1982;79:7522-6.
1985;89:252-8
Immunoglobulin M Antibody to Hepatitis B Core Antigen in Patients With Chronic Type B Hepatitis MARIA
SJOGREN and JAY H. HOOFNAGLE
Department of Virus Diseases, Walter Reed Army Institute of Research, Washington, DC. and the Liver Diseases Section, Digestive Diseases Branch, National Institute of Arthritis, Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland
Serum samples from 130 persons who were seropositive for hepatitis B surface antigen and who had various forms of accompanying liver disease were tested for immunoglobulin M (IgM) antibody to hepatitis B core antigen. In 99% of patients with hepatitis B e antigen-positive chronic type B hepatitis, IgM antibody to hepatitis B core antigen was present. This antibody was not present in “healthy” hepatitis B surf&e antigen carriers and was detectable in only 30% of patients with 6 hepatitis. Testing of serial sera from 38 patients with chronic type B hepatitis revealed that IgM antibody to hepatitis B core antigen persisted in patients who had evidence ofpersistent hepatitis B virus replication but ultimately disappeared in those patients who exhibited a sustained loss of serum markers of viral replication [hepatitis B virus deoxyribonucleic acid and deoxyribonucleic acid polymerase activityl. These findings suggest that the presence of IgM antibody to hepatitis B core antigen in chronic hepatitis B surface antigen carriers indicates an active immune response to persistent viral replication. Antibody responses during acute viral infections are characterized by an early immunoglobulin M (IgM) antibody response that gradually wanes as a subsequent immunoglobulin G (IgG) response arises. This pattern of antibody class responses has been shown
Received October 5, 1984. Accepted February 11, 1985. Address requests for reprints to: Jay H. Hoofnagle, M.D., Liver Diseases Section, Building 10, Room 4-D-52, National Institutes of Health, Bethesda, Maryladd 20205. The opinions and assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense. The authors thank Jeanne G. Waggoner and Lee Sosebee for technical assistance. 0 1965 by the American Gastroenterological Association 0016-5085/85/$3.30
to occur in almost all acute viral infections including the various forms of viral hepatitis-type A, type B, and 6 hepatitis (l-7). Testing for IgM antibody to specific viral antigens has been proposed as a serologic means of diagnosis of the type of viral hepatitis (Z-6).Less well characterized are the antibody class responses that occur during chronic viral infections. If viral replication becomes chronic, IgM antibodies to viral antigens frequently persist. The persistence of IgM antibody has been shown in both chronic type B and chronic &hepatitis infections (6-10)as well as in several other chronic viral diseases such as congenital rubella (ll),subacute sclerosing panencephalitis (l2), and chronic infectious mononucleosis (131.
While investigating the IgM antibody response to hepatitis B core antigen (IgM anti-HBc) among chronic hepatitis B surface antigen (HBsAg) carriers, we have found that this antibody response defines several features of the natural history of chronic type B hepatitis. Patients with persistence of viral replication and serum biochemical evidence of ongoing chronic hepatitis maintained detectable serum levels of IgM anti-HBc. Patients who underwent a remission in disease activity and who lost detectable markers of viral replication remained HBsAg-positive, but usually lost IgM anti-HBc. The presence of detectable IgM anti-HBc, therefore, was a sensitive marker of active viral replication and indicated that the accompanying liver disease was due to the hepatitis B virus (HBV) infection. The absence of this antibody indicated a lack of HBV replication and suggested that any accompanying hepatitis was due to a superimposed, unrelated or secondary form of
Abbreviations used in this paper: IgM, IgG, immunoglobulins M and G, respectively; IgM anti-HBc, immunoglobulin M antibody to hepatitis B core antigen; HBV-DNA, hepatitis B virusdeoxyribonucleic acid.
August 1985
IgM ANTI-HBc
liver disease (type A, non-A, non-B, or 6 hepatitis drug-induced liver injury].
or
Materials and Methods Patient Population The study comprised 130 patients who had HBsAg in serum and who were being followed at the National Institutes of Health Clinical Center. All 130 patients were known to have had HBsAg in serum for at least 6 mo before this serologic and clinical evaluation. Patients were classified into four groups. Group 1 consisted of 100 patients who were seropositive for the hepatitis B e antigen (HBeAg) and who had elevations in the serum alanine aminotransferase levels (ALT) [mean ALT 253, range 4% 1370 U/L]. Superinfection with the HBV-associated 6 agent was excluded in these patients by the absence of anti-3 in the serum. These 100 patients in group 1 were defined as having HBeAg-positive chronic type B hepatitis. Group 2 consisted of 10 patients who were seropositive for antibody to the hepatitis B e antigen (anti-HBe) and who had normal serum ALT levels (mean ALT 29, range 15.-36 U/L). These patients were defined as asymptomatic “healthy” HBsAg carriers without evidence of active liver disease. Group 3 consisted of 10 patients who were seropositive for anti-HBe but who, nevertheless, had elevated serum ALT levels (mean ALT 163, range 48-414 U/L). In 9 of these patients, a liver biopsy specimen was taken; all specimens revealed histologic evidence of chronic hepatitis. These patients were considered to have anti-HBe-positive chronic type B hepatitis. Group 4 consisted of 10 patients who were seropositive for antibody to the 6 agent (in titers > 1: 100) and who had elevated serum ALT levels [mean ALT 293, range 63-1065 U/L). These patients were defined as having chronic 6 hepatitis (7). To define the behavior of IgM anti-HBc during the course of chronic type B hepatitis, serial serum specimens from 38 of the patients from group 1 (HBeAg-positive) were tested for this antibody. These 38 patients have been followed for l-9 yr [mean 3.1 yr). The patients were routinely seen every l-6 mo and had blood tested for serologic markers of HBV infection as well as routine laboratory biochemical tests including ALT, aspartate aminotransferase, bilirubin, albumin, and globulin using automated techniques (SMAC: Technicon Instruments Corp. Tarrytown, N.Y.). Serologic
Testing
Serum specimens were tested for HBsAg and antibody (anti-HBs) by radioimmunoassays (Ausria-II and Ausab: Abbott Laboratories, North Chicago, Ill.). Serum HBeAg and anti-HBe were assayed by immunodiffusion and radioimmunoassay (HBeAg Test, Abbott) (14,15). Serum hepatitis B virus-deoxyribonucleic acid (HBV-DNA) was measured by molecular hybridization using a 32Plabeled HBV-DNA probe [Bethesda Research Labs, Bethesda, Md.) (16). Hepatitis B virus-DNA polymerase activity was assayed by determining [3H]thymidine incorporation (17). A single specimen from each patient was also tested for antibody to the 6 agent (anti-61 by solid-phase radio-
253
immunoassay (courtesy of Dr. John Gerin and Ronald Engle) (7). Immunoglobulin M antibody to hepatitis B core antigen was assayed by a solid-phase, antibody-capture radioimmunoassay technique (3). Polyvinyl microtiter plates were coated with goat antihuman IgM (w-chain specific]. Fifty microliters of serum diluted 1:100 in phosphatebuffered saline was added to the microtiter wells and incubated for 4 h at 30°C. The plates were washed with phosphate-buffered saline and 25 ~1 of a preparation of hepatitis B core antigen purified from chimpanzee liver was added. The microtiter plates were incubated overnight and then washed. Finally, 40 ~1 of ‘251-labeled human IgG anti-HBc (200,000 cpm) was added to each well and left for 4 h at 4°C. The plates were washed again, the wells cut out, and the radioactivity adhering to the microtiter wells was counted in a y-counter. Controls included specimens from normal individuals without HBV infection. Samples were also tested without the addition of purified hepatitis B core antigen so as to detect rheumatoid-factor-like activity that might yield false-positive results. The radioimmunoassay ratio of sample counts per minute to negative control mean counts per minute was used as a measure of IgM anti-HBc level. Negative samples yielded radioimmunoassay ratios of ~2.1, whereas strongly positive samples, such as those from patients with acute type B hepatitis, yielded ratios of >15 (3). Single samples from each patient were also tested for IgM anti-HBc using a commercial enzyme immunosorbent assay (Corzyme-M, Abbott) in which samples are assayed at a dilution of -1:lOOO (4). Percutaneous liver biopsies had been performed on 93 of these 130 patients, including 78 from group 1, none from group 2, 9 from group 3, and 6 from group 4. The biopsy specimens were categorized by standard histologic criteria as either chronic persistent hepatitis, chronic active hepatitis, or cirrhosis (18).
Statistical
Analyses
Group frequencies were compared using the x2 test. Values within groups were compared by means of correlation coefficient analysis. Changes in laboratory test results in individual patients were analyzed by the paired Student’s t-test.
Results Results of testing sera for IgM anti-HBc in the four groups of patients are shown in Table 1. All except 1 patient with HBeAg-positive chronic type B hepatitis were seropositive ,for IgM anti-HBc by radioimmunoassay. The single exception was an asymptomatic Asian man who had been known to be HBsAg-positive for many years and whose serum ALT levels were persistently less than twice the upper limit of the normal range. Of the 100 HBeAgpositive patients, 99 had detectable HBV-DNA in serum and 97 had DNA polymerase activity.
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Table
1. Results of Testing Four Groups of Hepatitis B Surface Antigen-Positive Patients for lmmunoglobulin M Antibody to Hepatitis B Core Antigen by Radioimmunoassay No. nositive
or test results for IgM antiHBc
Group HBeAg+ chronic type B Healthy HBsAg+ carriers Anti-HBe+ chronic type B 6 Hepatitis
No. tested
HBeAg+
HBV-DNA+ or DNAp+
No. +
Mean level”
100
100
99
99
6.2
10
0
0
1
1.7
10
0
5b
10
9.0
10
3
3
3
2.7
HBsAg, hepatitis B surface antigen; anti-HBc, antibody to hepatitis B core antigen; HBeAg, hepatitis B e antigen; anti-HBe, antibody to HBeAg; HBV-DNA, hepatitis B virus-deoxyribonucleic acid; DNAp, DNA polymerase activity. ” Level expressed as radioimmunoassay ratio units = sample counts per minute/ negative control mean counts per minute: >2.1 is considered positive. b Intermittently positive.
The HBsAg-positive patients who were negative for fiBeAg were less likely to have detectable IgM anti-HBc. Among 10 patients who were considered to be healthy HBsAg carriers without active liver disease (group 2), only 1 was weakly seropositive for this IgM antibody. None had detectable HBV-DNA or DNA polymerase in serum. In contrast, the 10 patients with anti-HBe who had elevated ALT levels (group 3) were all positive for IgM anti-HBc, often at high levels. Five of these patients were intermittently positive for HBV-DNA and DNA polymerase at low levels. The 10 patients with 6 hepatitis (group 4) were Table
2. Correlations Between Immunoglobulin Evidence of Chronic Hepatitis Disease
IgM anti-HBc radioimmunoassay ratio results”
No.
Negative (<2.1)
10
usually negative for IgM anti-HBc. Three patients had low levels of IgM anti-HBc. Although sera from a total of 113 of the 130 patients were reactive for IgM anti-HBc using radioimmunoassay, sera from only 2 were reactive for this IgM antibody using the commercial enzyme-linked immunosorbent assay. These 2 patients were in the midst of a severe exacerbation of their disease at the time. Both patients had serum ALT levels >800 u/L and 1 patient was jaundiced at the time that they were seroreactive for this antibody. Both patients eventually improved and this reactivity disappeared. The level of IgM anti-HBc as detected by radioimmunoassay correlated to spme extent with the severity and the activity of the underlying chronic type B hepatitis. Thus, the mean of radioimmunoassay ratio units in patients with cirrhosis was higher (mean f SD = 9.7 ‘_ 4.6) than in those patients with chronic persistent hepatitis (6.9 t 2.8; p < 0.05) or chronic active hepatitis (7.3 ? 3.1; p < 0.05). In addition, there was a positive correlation between the height of serum ALT and aspartate aminotransferase levels and the level of IgM aptiHBc (Table 2). This correlation between IgM antiHBc and serum biochemical evidence of activity of liver disease was most marked among HBsAg-positive patients with anti-HBe. Furthermore, this correlation did not hold true for patients with S hepatitis; many of the patients with 6 hepatitis had cirrhosis (70%) and marked elevations in serum ALT and aspartate aminotransferase levels, yet these 10 patients were either seronegative for IgM anti-HBc or had low levels of the antibody. Finally, although there was a correlation between the presence of IgM anti-HBc and the presence of HBV-DNA and DNA
M Antibody to Hepatitis Activity in 120 Hepatitis
B Core Antigen Levels and Biochemical B Surface Antigen-Positive Patients
Mean (SD] of serum ALT (U/L]
AST (U/L)
Albumin (gldl)
Protime (s)
4.5
11.6
(1;) 117
(0.2) 4.3
CO.51 12.1
2.1-4.9
25
(ii, 239
5.0-9.9
63
(2411 218
(167) 111
(0.31 4.0
(0.9) 13.3
10.0-14.9
17
(1961 275
(136) 137
(0.4) 4.0
(1.2) 12.9
5
(2271 498
(115) 250
(0.4) 3.8
(1.51 13.6
(0.41
Cl.41
>15.0
(3611
Correlation coefficient p Value
Vol. 89, No. 2
(151)
0.261
0.226
-0.387
co.02
0.325
DNAP (cpm) 129 (261) 2796 (2714) 2367 (2881) 1655 (1392) 2253 (2611) 0.014
(r)b <0.005
NS
Anti-HBc, antibody to hepatitis B core antigen; ALT, alanine aminqtransferase; AST, aspartate aminotransferase: protime, prothrombin time; DNAp, DNA polymerase; cpm, counts per minute; NS, not significant or p > 0.05. a Radioimmunoassay ratio = sample counts per ratio minute/negative control counts per minute: >2.1 is positive. b Correlation coefficient between the IgM anti-HBc radioimmunoassay units and the biochemical laboratory result analyzed.
August
1985
IgM ANTI-HBc
Table 3. Serologic Markers in
38
255
Patients With Chronic Type B Hepatitis at the Start and End of Follow-up
Evaluation IgM Group Persistent
(No.]
Time
HBeAg
ALT” (U/L)
DNAp”
HBeAg
Start End Start End Start End
+ + + _
125 (239) 183(?164) 446 (2313) 35 (211)" 150 (267) 123 (263)
3558(+1860) 4459(?3673) 3573(?4037) 28 (211)" 2014 (22833) 1578(t3390)
(20) Seroconversion (13) Reactivation (51
+ +
(cpm)
anti-HBc” (RIA ratio) 7.9 (k3.0) 8.2 (k3.5) 10.3 (24.5) 1.9 (21.3)" 9.4 (25.2) 7.9 (22.8)
HBeAg, hepatitis B e antigen; ALT, alanine aminotransferase activity (normal ~45 U/L); DNAp, DNA polymerase (negative
polymerase in serum, there was no correlation between the level of IgM anti-HBc and the level of the HBV markers in serum. Serial specimens from 38 patients with chronic HBeAg-positive hepatitis were tested for IgM antiHBc by radioimmunoassay. These patients were selected to represent three different outcomes of HBeAg-positive chronic type B hepatitis (14). Twenty patients remained HBeAg-positive and continued to have elevated serum ALT levels (persistent HBeAg). In all 20 of these patients, IgM anti-HBc remained detectable in the serum and titers did not change appreciably (Table 3 and Figure 1).Thirteen of the 38 patients became HBeAg-negative and seroconverted to anti-HBe during the period of follow-up evaluation (HBeAg seroconversion). Associated with
this seroconversion, serum HBV-DNA and DNA polymerase activity became undetectable and serum ALT levels decreased. The serum levels of IgM antiHBc fell in all 13 patients and became undetectable in 12 (Figure 2). In these patients, the fall in serum IgM anti-HBc into the negative range occurred 0.5-5 yr (mean 1.9 yr) after the disappearance of HBeAg. The remaining 5 patients became HBeAg-negative, but serum ALT levels improved only temporarily. These 5 patients exhibited a return of biochemical evidence of disease concurrent with a return of HBVDNA and DNA polymerase in serum (spontaneous reactivation of chronic type B hepatitis) (19).Serum IgM anti-HBc reactivity remained present in all 5 patients and levels of this antibody usually increased during the period of exacerbation (Figure 3).
3 3 a
200
ALT
150
DNA Polymerase
1979
1980
1981
1982
1983
YEAR Figure
1
Serum biochemical and serologic course of a patient with chronic type B hepatitis who remained hepatitis B e antigen (HBeAg)-positive for 5 yr. Serum alanine aminotransferase levels (ALT) were persistently elevated and DNA polymerase activity and immunoglobulin M antibody to hepatitis B core antigen (IgM anti-HBc) remained present. cpm, counts per minute; RIA ratio, radioimmunoassay ratio.
256
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1991
1992
Vol. 89, No. 2
1983
YEAR Figure
2. Serum biochemical and serologic course of a patient with chronic type B hepatitis who became seronegative for hepatitis B e antigen (HBeAg) and developed antibody to HBeAg (anti-HBe) during the period of observation. Serum DNA polymerase activity became negative immediately before HBeAg became undetectable and, shortly thereafter, serum alanine aminotransferase (ALT) levels fell into the normal range. Serum immunoglobulin M antibody to hepatitis B core antigen (IgM anti-HBc) levels gradually decreased, becoming undetectable 18 mo after HBeAg became negative.
T
nIgM
Anti-HBc
DNA / Polymerase
I _J lo
Boo 400
5
200 2
100 1980
1981
1982
1983
YEAR Figure
3. Serum biochemical and serologic course of a patient with chronic type B hepatitis who became seronegative for hepatitis B e antigen (HBeAg) and developed antibody to hepatitis B e antigen (anti-HBe) and concurrently had an improvement in serum aminotransferase (ALT) levels. With the loss of HBeAg, serum levels of immunoglobulin M antibody to hepatitis B core antigen (IgM anti-HBc) began to fall. Subsequently, however, this patient had a reactivation of disease with return of biochemical evidence of chronic hepatitis and serologic evidence of viral replication (DNA polymerase activity and HBeAg). This spontaneous exacerbation of disease was accompanied by a marked rise in IgM anti-HBc.
August
1985
Discussion The results of this study indicate that the majority of patients with chronic type B hepatitis continue to produce detectable amounts of IgM antibody to the core component of the HBV. This antibody was found in 99% of patients who had HBeAg detectable in serum and in all of a small number of patients who were seronegative for HBeAg but who, nevertheless, appeared to have continued chronic type B hepatitis. These findings suggest that the continued presence of hepatitis B core antigen induces a continued production of specific IgM antibody. Previous reports have proposed that IgM anti-HBc could be used as a serologic marker for acute type B hepatitis (3-6).During acute infection, levels of IgM anti-HBc are usually quite high, whereas levels of IgG anti-HBc are low. With resolution of the disease, levels of IgM anti-HBc begin to fall, and this antibody is usually undetectable 3-24 mo later. In the present study, IgM anti-HBc was shown to persist in patients with chronic HBV infection, a finding that would seem to obviate the use of this antibody as a reliable marker for acute infection. However, the radioimmunoassay used in the current study differed in several important details from those immunoassays for IgM anti-HBc that are used to make the diagnosis of acute type B hepatitis. Thus, the commercial enzyme-linked immunosorbent assay was designed to only detect high titers of IgM anti-HBc such as occur during acute type B hepatitis. The assay uses a high dilution of test sample (-1: 1000) and concentrations of reagent antigen and antibody that facilitate detection of high and not low levels of IgM anti-HBc. In contrast, the radioimmunoassay used in the current study was designed to be a sensitive assay for IgM anti-HBc using the concentrations of anti-IgM and hepatitis B core antigen and the dilution of serum (1:100) that yield maximal sensitivity and specificity (3). For these reasons, these two assays are not easily comparable. Furthermore, the commercial immunosorbent assay cannot be readily adapted for the detection of IgM anti-HBc that occurs in chronic type B hepatitis. The finding of moderate or low titers of IgM antiHBc in patients with chronic type B hepatitis has been previously reported by several groups of investigators using a variety of immunologic assays for this antibody (5,6,8-10). This IgM antibody has been shown to differ from that found during acute type B hepatitis in that it is often 7-8s IgM rather than the typical 1% antibody (20,21). The current study differed from previous studies in that the IgM antiHBc was assayed not just in single serum samples but also in serial serum specimens from patients with chronic type B hepatitis. This allowed for
IgM ANTI-HBc
257
analysis of the correlation between changes in IgM anti-HBc levels and changes in biochemical and serologic features of the disease. This testing of sequential samples demonstrated that this antibody persisted in the serum of patients with persistent viral replication (continued presence of HBeAg, HBV-DNA, and DNA polymerase); but that it disappeared from the serum of those persons who lost serologic evidence of active viral replication even though these patients remained positive for HBsAg. Thus, persistence of IgM anti-HBc in the serum appeared to correlate with serologic evidence of continued viral replication. Several serologic tests have been proposed as sensitive markers of active HBV replication. Chief among these has been HBeAg, the presence of which correlates well with high levels of HBV in serum and high degrees of infectivity (14).However, HBeAg is not present in the serum of all patients who continue to produce HBV particles. Other investigators have suggested that HBV-DNA or DNA polymerase may be more direct and perhaps more sensitive markers of the production of HBV (16,17,19,22,23). Indeed, using these two serologic tests one can sometimes detect HBV in serum that contains anti-HBe and no detectable HBeAg. As shown here and by other investigators, however, even these direct markers of HBV production are not invariably present when there is serum biochemical and hepatic histologic evidence of continued chronic type B hepatitis activity (23-25).In this situation, however, IgM anti-HBc is usually detectable. In the current study, IgM antiHBc was found in all patients in a group of 10 who did not have HBeAg in serum but who did have clinical, serum biochemical, and histologic evidence of chronic hepatitis. In this same group, HBV-DNA and DNA polymerase activity were present in the serum of some patients, but only intermittently and at low titer. The presence of IgM anti-HBc, therefore, appeared to mirror the presence of even low levels of HBV production. Interestingly and importantly, however, the level of IgM anti-HBc did not correlate with the titer of HBV produced (as quantified by the level of HBV-DNA or DNA polymerase in serum). Instead, the level of IgM anti-HBc correlated better with histologic and biochemical evidence of active liver disease (10). Levels of IgM anti-HBc were highest in patients with active liver disease and were absent or low in patients who were healthy carriers or who had mild forms of chronic type B hepatitis. Testing of sequential serum specimens indicated that remissions in the chronic liver disease were accompanied by decreases in level of IgM anti-HBc. Typically, IgM anti-HBc fell to undetectable levels between 12 and 24 mo after the chronic hepatitis resolved. This
258
SJOGREN
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HOOFNAGLE
delay in disappearance of IgM anti-HBc after improvement in chronic type B hepatitis is another reason why testing of single serum specimens may yield conflicting results. The delay in fall of specific IgM anti-HBc after disappearance of active viral replication merely reflects the delay in disappearance of antibody responses after antigen challenge. From these data, one could hypothesize that the presence of IgM anti-HBc reflects both the presence of continued viral replication as well as an immune response to that replication. Accordingly, patients who produce high titers of virus but who have little in the way of an immune response to the virus would tend to have low or mediocre titers of IgM anti-HBc. In contrast, patients who have an active, accompanying immune response to the viral replication would tend to have high levels of IgM anti-HBc. This interpretation also would afford an explanation for the high titers of IgM anti-HBc that are found during acute viral hepatitis in which the immune response to the virus is typically strong and usually leads to complete elimination of the viral infection. Thus, IgM anti-HBc can be used as a serologic marker to indicate the presence of continued HBV replication as well as an immune response to the virus. The detection of this antibody is especially helpful in patients who have no detectable HBeAg in serum but who do have active accompanying liver disease. If the liver disease is due to HBV, IgM antiHBc should be present. If the liver disease in the HBsAg-positive and anti-HBe-positive patient is not due to HBV but is due to a superimposed and unrelated form of liver disease (such as S hepatitis or perhaps drug-induced liver injury], IgM anti-HBc should be absent.
References 1. Locarnini SA, Ferris AA, Lehmann NI, Gust ID. The antibody response following hepatitis A infection. Intervirology 1977; 8:309-18. 2. Decker RH, Kosakowski SM, Vanderbilt AS, Ling CM, Chairez R, Overby LR. Diagnosis of acute hepatitis A by HAVAB-M, a direct radioimmunoassay for IgM anti-HAV. Am J Clin Path01 1981;76:140-7. 3. Lemon SM, Gates NL, Simms TE, Bancroft WH. IgM antibody to hepatitis B core antigen as a diagnostic parameter of acute infection with hepatitis B virus. J Infect Dis 1981;143:803-9. 4. Chau KH, Hargie MP, Decker RH, Mushahwar IK, Overby LR. Serodiagnosis of recent hepatitis B infection by IgM class anti-HBc. Hepatology 1983;3:142-9. 5. Kryger P, Mathiesen LR, Aldershvile J, Nielsen JO, The Copenhagen Hepatitis Acuta Programme. Presence and meaning of anti-HBc IgM as determined by ELISA in patients with acute type B hepatitis and healthy HBsAg carriers. Hepatology 1981;1:233-7. 6. Shimizu M, Ohyama M, Takahaski Y, et al. Immunoglobulin M antibody against hepatitis B core antigen for the diagnosis
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of fulminant type B hepatitis. Gastroenterology 1983;84:60410. 7. Smedile A, Lavarini C, Crivelli 0, et al. Radioimmunoassay detection of IgM antibodies to the HBV-associated delta (8) antigen. Clinical significance of 8 infection. J Med Virol 1982;9:131-8. 8. Lemon SM, Hoofnagle JH. IgM antibody to hepatitis B core antigen in patients with chronic type B hepatitis. In: Szmuness W, Alter HJ, Maynard JE, eds. Viral hepatitis. 1981 International Symposium. Philadelphia: Franklin Institute Press, 1981:723-4. 9. Hoofnagle JH. Specific IgM and IgG antibody responses during type B hepatitis. In: Berk PD, Chalmers TC, eds. Frontiers in liver disease. New York: Thieme-Stratton, 1981:208-12. 10. Banninger P, Altorfer J, Froesner GG, et al. Prevalence and significance of anti-HBc IgM (radioimmunoassay) in acute and chronic hepatitis B and in blood donors. Hepatology 1983;3:337-42. 11. Alford CA. Studies on antibody in congenital rubella infections. Am J Dis Child 1965;110:455-63. 12. Kiessling WR, Hall WW, Yung LL, Ter Meuten V. Measlesvirus specific immunoglobulin M response in subacute sclerosing panencephalitis. Lancet 1977;i:324-7. 13. Tobi M, Morag A, Ravid Z, et al. Prolonged atypical illness associated with serological evidence of persistent EpsteinBarr virus infection. Lancet 1982;i:61-4. 14. Hoofnagle JH, Dusheiko GM, Seeff LB, Bales ZB, Waggoner JG, Jones EA. Seroconversion from hepatitis B e antigen to antibody in chronic type B hepatitis. Ann Intern Med 1981; 94:744-8. 15. Krugman S, Overby LR, Mushahwar IL, Ling CM, Froesner GG, Dienhardt F. Viral hepatitis, type B. Studies on natural history and prevention re-examined. N Engl J Med 1979; 300:101-7. 16. Berninger M, Hammer M, Hoyer B, Gerin JL. An assay for the detection of the DNA genome of hepatitis B virus in serum. J Med Virol 1982;9:57-68. 17. Kaplan PM, Greenman RL, Gerin JL, Purcell RH, Robinson WS. DNA polymerase associated with human hepatitis B antigen. J Virol 1973;12:995-1005. 18. International Group. Acute and chronic hepatitis revisited. Lancet 1977;ii:914-9. 19. Davis GL, Hoofnagle JH, Waggoner JG. Spontaneous reactivation of chronic type B hepatitis. Gastroenterology 1984;86: 230-5. 20. Sjogren M, Lemon SM. Low molecular weight IgM antibody to hepatitis B core antigen in chronic hepatitis B virus infections. J Infect Dis 1983;148:445-51. 21. Tsuda F, Naito S, Takai E, et al. Low molecular weight (7s) immunoglobulin M antibody against hepatitis B core antigen in the serum for differentiating acute from persistent hepatitis B virus infection. Gastroenterology 1984;87:159-64. 22. Bonino F, Hoyer B, Nelson J, Engle R, Verme G, Gerin JL. Hepatitis B virus DNA in the sera of HBsAg carriers. A marker of active hepatitis B virus replication in the liver. Hepatology 1981;1:386-91. 23. Hadziyannis SJ, Lieberman HM, Karvountzis GG, Shafritz DA. Analysis of liver disease, nuclear HBcAg, viral replication, and hepatitis B virus DNA in liver and serum of HBeAg vs. anti-HBe positive carriers of hepatitis B virus. Hepatology 1983;3:656-62. 24. Alberti A, Tremolada F, Fattovich G, Bortolotti F, Realdi G. Virus replication and liver disease in chronic hepatitis B virus infection. Dig Dig Sci 1983;28:962-6. 25. Kam W, Rall LB, Smuckler EA, Schmid R, Rutter WJ. Hepatitis B viral DNA in liver and serum of asymptomatic carriers. Proc Nat1 Acad Sci USA 1982;79:7522-6.