THE JOURNAL OF Il'\FECTIOUS DISEASES. VOL. 135, NO.6. JUNE 1977 © 1977 by the University of Chicago. All rights reserved.

Antibodies. to Hepatitis B Core Antigen in Hepatitis B Surface Antigen-Positive and -Negative Chronic Hepatitis Michael A. Gerber, Teresa Zappi, Salvatore J. Vernace, and Fiorenzo Paronetto

From the Department of Pathology, and the Division of Liver Disease of the Department of Medicine, Mount Sinai School of Medicine of The City University of New York, New York, New York; and the Laboratory Service, Veterans Administration Hospital, Bronx, New York

Antibody "to hepatitis B core antigen (HB c Ag) has been recognized as a sensitive indicator of current or recent infection with hepatitis B virus (HBV) [1]. Many techniques have been described for the detection of antibody to HB c Ag (anti-HB c), including immunodiffusion [2], counterimmunoelectrophoresis [3], indirect fluorescent antibody technique [4, 5], immunoagglutination electron microscopy [6], CF [7], immune adherence HA [8], micro-solid-phase-radioimmunoassay [9], and radioimmunoprecipitation of core particles radiolabeled by DNA polymerase reaction [10, 11]. Each of these tests has inherent advantages and disadvantages; the major drawback of all tests is the lack of sufficient quantities of purified HB c Ag, since HBV continues to elude efforts directed at propagation in tissue culture [12]. However, the results of several studies employing different methods were similar in that anti-HB c was regularly detected in patients with HBV infection, i.e., chronic carriers [5, 9, Received for publication September 14, 1976, and in revised form December 9,1976. Dr. Gerber is the recipient of Research Career Development Award no. I K04 Al 00035 from the U.S. Public Health Service. Please address requests for reprints to Dr. Michael A. Gerber, Department of Pathology, Mount Sinai School of Medicine, Fifth Avenue and looth Street, New York, New York 10029.


13, 14], patients with acute hepatitis B [5, 9, 1316], or patients with hepatocellular carcinoma [3]. Except for one report [5], patients with chronic hepatitis have not been studied in detail for markers (including anti-HB c) of HBV infection. Therefore, we embarked on such a study of patients with chronic hepatitis, including both those whose sera contained hepatitis B surface antigen (HB s Ag) (seropositive) and those whose sera did not contain this antigen (seronegative). Materials and Methods

Sixty-seven patients with clinical and histological diagnosis of chronic hepatitis, with or without cirrhosis, were selected for this study. Selection was based on the availability of serum specimens. The patients, 22 females and 45 males, ranged in age from 17 to 78 years and were followed prospectively by one of us (S.J.V.). Detailed clinical and biochemical data and at least one liver biopsy specimen were available for all patients. A total of 124 sera were tested. Two or more samples of serum were studied from 40 patients. The first of these serial specimens and the only specimen from the remaining 27 patients were obtained prior to treatment. All sera were stored at -20 C. The following tests were performed. (1) HB s

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Antibody to hepatitis B core antigen (anti-HB c) is a sensitive indicator of hepatitis B virus (HBV) infection. However, anti-HB c has been found in only a few patients with chronic hepatitis. Therefore, we tested for anti-HB c in 124 sera from 67 patients with histologically proven chronic hepatitis by the indirect fluorescent antibody technique. All patients, except for one with chronic hepatitis who was seropositive for hepatitis B surface antigen (HB s Ag), had anti-HB c that persisted throughout the follow-up period (three months to three years). Of 33 HB s Ag-seronegative patients, anti-HB c was detected in seven patients and persisted for six months to two years. These findings suggest that in this study 21 % of patients with chronic hepatitis with undetectable amounts of HB s Ag in the serum had evidence of recent or continued HBV replication.

Anti-HB c in Chronic Hepatitis

Table 1. Incidence of antibody to hepatitis B core antigen (anti-HB c ) in sera of patients with chronic hepatitis. Serum

No. of sera

No. positive for anti-HB c (%)

55 69

18 (26)

HB s Ag-positive HB s Ag-negative

50 (91)

NOTE. HB s Ag == hepatitis B surface antigen.

of both HB c Ag-positive and normal human liver in a homogeneous or peripheral pattern. In addition, sera with both ANA and anti-HB c and sera HB s Ag-negative and anti-HBc-positive were absorbed one to three times with an equal volume of purified normal human liver nuclei (concentration of protein, 100 mg/ml) [19]. Three sera with ANA were used as controls for the efficiency of the absorption. After absorption the sera were retested on glycine-eluted normal human liver and on HB c Ag-positive liver. Results

Of the 124 sera tested, 55 were HB s Ag-positive and 69 were HB s Ag-negative. Of these sera, 91 % and 26%, respectively, had anti-HB c (table 1). In terms of the number of patients (table 2), anti-HB c was detected in all but one of 34HB s Ag-seropositive patients. In this patient, a 32year-old man with high titers of MA, tests for anti-HB c were negative in two RB s Ag-positive specimens of serum taken five months apart. In one patient, anti-HB c became negative simultaneously with HB s Ag after both had been present in three specimens obtained over a period of two and one-half years. In another patient, a postmortem sample of serum was negative for anti-RBc six weeks after the same individual was seropositive for anti-HB c, while HB s Ag was deTable 2. Serological findings in 67 patients with chronic hepatitis.

Serum HB s Ag-positive HB s Ag-negative

No. positive for

No. of patients

Anti-HB c

Anti-HB s

34 33

33 7

4 7

NOTE. HB s Ag == hepatitis B surface antigen; anti-HB c == antibody to hepatitis B core antigen; anti-HBs == antibody to HB s Ag.

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Ag was measured by solid-phase radioimmunoassay (Ausria II, Abbott Laboratories, North Chicago, Ill.); (2) anti-HB s (antibody to HB s Ag) subtypes ad and ay by passive HA (Virgo Laboratories, Bethesda, Md.); (3) anti-HB c by indirect immunofluorescence [5]; and (4) antibodies to nuclei (ANA), mitochondria (MA), and smooth muscle (SMA) by the indirect fluorescent antibody technique with normal human liver and kidney and rat stomach and kidney as substrates. Sera with a positive reaction at a dilution of 1: 10 were considered positive and were serially diluted [5]. For the determination of anti-HB c by the indirect fluorescent antibody technique, human liver containing many 27-nm core particles in approximately 50% of hepatocellular nuclei, as determined by electron microscopy, served as substrate. The core particles had been shown by immune electron microscopy to react with anti-HB c [17]. Immunoglobulin bound in vivo [18] was eluted from frozen sections of the HB c Ag-containing liver with 0.1 M glycine-HCl buffer (pH 2.2) before treatment with patients' sera; this step was followed by reaction with fluorescein isothiocyanate-conjugated polyvalent rabbit antiserum to human y-globulin. The antiserum had been prepared by immunization of rabbits with Cohn fraction II of human plasma (E. R. Squibb and Sons, New York, N.Y.) and, by the direct fluorescent antibody technique, it reacted with myelomatous tissue derived from patients with IgG and IgA myelomas and with Waldenstrom's macroglobulinemia. Complete elution of bound immunoglobulin without loss of intranuclear HB c Ag was determined by direct staining of liver sections with fluorescein isothiocyanate-conjugated rabbit antiserum to human y-globulin and anti-HB c. Furthermore, normal human sera and an anti-HBc-containing reference serum were used as controls. To exclude specimens that were falsely positive for anti-HB c because of ANA or nonspecific binding to liver nuclei, all sera were tested on glycineeluted normal human liver. Activity of ANA could be distinguished easily from anti-HB c because anti-HB c reacted with 50% of hepatocellular nuclei of the HB c Ag-positive liver in a granular pattern, whereas ANA reacted with all hepatocellular, reticuloendothelial, and bile duct nuclei




Almost all HB s Ag-seropositive patients with chronic hepatitis had anti-HB!, that persisted for the entire follow-up period of up to three years. Similar findings have been reported for chronic

carriers of HB s Ag [5, 9, 13, 14], although only one study included 20 patients with histologically proven chronic hepatitis [5]. More importantly, however, anti-HB e was detected in seven of 33 HB s Ag-seronegative patients with chronic hepatitis or in 26~~ of serum specimens that were negative for HB s Ag by radioimmunoassay. Hoofnagle et al. [14] described a gap of several weeks' duration between transient HB s Ag reactivity and the appearance of anti-HBs in patients with acute viral hepatitis B; during this interval the serum was positive for anti-HB e only [l4J. This gap might also occur in patients with chronic hepatitis who" during the initial period of HB s antigenemia, might not be tested for HB s Ag because of the insidious onset of the disease. More likely, however, these HB s Ag-seronegative, anti-HB e seropositive patients with chronic hepatitis are comparable to blood donors implicated in cases of posttransfusion hepatitis who were positive for anti-HB e only [1]. The presence of anti-H~e in the serum of such patients may be a sensitive indicator of persistent viral replication when undetectable amounts of HB s Ag are circulated. The detection of HB s Ag or HB e Ag in the livers of these patients would support this hypothesis. Indeed, HB e Ag, but not HB s Ag, has been detected by the direct fluorescent antibody technique in 30% of hepatocellular nuclei of an HB s Ag-seronegative, anti-HBe-seropositive patient, from whom a frozen liver biopsy specimen was available. In chronic hepatitis, the presence of antiHB e may identify HB s Ag-seronegative patients in whom the disease is nevertheless related to HBV infection. This finding might be important for the prognosis and therapy of chronic hepatitis, as well as for the differential diagnosis of chronic hepatitis, primary biliary cirrhosis, and cases with overlap of chronic hepatitis and primary biliary cirrhosis [20]. Therefore, antiHB e is an important and sensitive marker of HBV infection and is helpful in the evaluation and understanding of chronic hepatitis. All presently detectable HBV markers together, i.e., HB s Ag, anti-HB e, and anti-HBs, distinguish a subgroup of chronic hepatitis that may be related to HBV infection. In this chronic viral hepatitis, autoimmune markers are generally absent from the serum. In contrast, chronic

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tected in both sera. In all other HB s Ag-seropositive patients, anti-HB e persisted throughout the follow-up period (three months to three years). One HB s Ag-seropositive patient lacked anti-HB e in the initial serum sample but was reactive three years later. Of 33 HB s Ag-seronegative patients, anti-HB e was detected in seven. In four of these patients, two or more specimens were studied, and the anti-HB e persisted throughout the follow-up period (six months to two years), whereas tests for HB s Ag were always negative. All but one of the seven anti-HBe-seropositive patients (two females and five males) had received blood transfusions or were drug addicts. Absorption of these sera with normal human liver nuclei did not diminish the staining of HB e Ag in liver nuclei. Anti-HB s was detected in four HB s Ag-seropositive and in seven HB s Ag-seronegative patients (table 2). The subtypes were anti-d in nine patients and anti-y in two patients. Of the seven patients who were HB s Ag-seronegative and antiHBe-seropositive, five had anti-HBs' The autoimmune markers (ANA, MA, SMA) were detected in 14 of 67 patients with chronic hepatitis. Ten of these patients were negative for all markers of current or past HBV infection (HB s Ag, anti-HB e, anti-HB s), and the autoantibody titer was> 1:80 in nine of them. Six patients had ANA, in three of these together with MA, and in one with SMA; two had SMA and two had MA only. Two of the four HB s Ag-seropositive patients had ANA and MA, respectively, at titers of ~1 :80, and two had SMA at titers of 1: 10 and 1:20. Absorption of the HBs-Ag positive, ANA-positive serum with normal human liver nuclei eliminated the reactivity with normal liver nuclei, but not that with hepatocellular nuclei of HB e Ag-positive liver. Of the 53 patients with chronic hepatitis who did not have autoantibodies, 39 were seropositive for HBV markers.

Gerber et al.

Anti-RB e in Chronic Hepatitis

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B core antigen by means of immune adherence hemagglutination. J. Immunol. 115:834-838,1975. Purcell, R. G., Gerin, J. L., Almeida, J. B., Holland, P. V. Radioimmunoassay for the detection of the core of the Dane particle and antibody to it. Intervirology 2:231-243,1973/74. Robinson, W .. S., Greenman, R. L. DNA polymerase in the core of the human hepatitis B virus candidate. J. Virol. 13:1231-1236, 1974. ~oritsugu, Y., Gold, J. W. ~., Wagner, J., Dodd, R. Y., Purcell, R. H. Hepatitis B core antigen: detection of antibody by radioimmunoprecipitation. J. Immuno!. 114:1792-1798, 1975. Zuckerman, A. J. Tissue and organ culture studies of hepatitis B virus. Am. J. ~ed. Sci. 270:197-204, 1975. Greenman, R. L., Robinson, W. S., Vyas, G. N. A sensitive test for antibody against the hepatitis B core antigen (anti-HB c)' Vox. Sang. 29:77-80, 1975. Hoofnagle, J. H., Gerety, R. ]., Barker, L. F. Antibody to hepatitis B core antigen. Am. J. ~ed. Sci. 270: 179-187,1975. Krugman, S., Hoofnagle, J. H., Gerety, R. J., Kaplan, P. ~., Gerin, J. L. Viral heptatitis, type B: DNA polymerase activity and antibody to hepatitis B core antigen. N. Engl. J. ~ed. 290:1331-1335,1974. Hollinger, F. B., Dreesman, G. R., Fields, H., ~elnick, J. L. HB c Ag, anti-HB c ' and DNA polymerase activity in transfused recipients followed prospectively. Am. J. ~ed. Sci. 270:343-348, 1975. Gerber, ~. A., Schaffner, F., Paronetto, F. Immunoelectron microscopy of hepatitis B antigen in liver. Proc. Soc. Exp. BioI. Med. 140:1334-1339, 1972. Gerber,~. A., Sarno, E., Vernace, S. J. Immune complexes in hepatocytic nuclei of HB Ag-positive chronic hepatitis. N. Engi. J. ~ed. 294:922-925, 1976. Hirschman, S. Z., Gerber, ~. A., Garfinkel, E. Purification of naked intranuclear particles from human liver infected by hepatitis B virus. Proc. Natl. Acad. Sci. U.S.A. 71 :3345-3349,1974. Popper, H., Gerber, M. A., Vernace, S. Immunologic factors in liver disease. lsI'. J. ~ed. Sci. 9:103-113, 1973. Villarejos, V. ~., Visona, K. A., Eduarte A., C. A., Provost, P. j., Hilleman, ~. R. Evidence for viral hepatitis other than type A or type B among persons in Costa Rica. N. Engl. J. ~ed. 293:1350-1352, 1975.

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hepatitis with autoimmune features is characterized by high titers of ANA, SMA, or MA and by the absence ~f RBV markers. A third subgroup, chronic hepatitis without RBV or autoimmune markers, still awaits clarification and may be related to non-A, non-B hepatitis virus infection [21]. More sensitive tests for anti-RBc may detect additional anti-RBc-seropositive cases among the third subgroup; finding of such additional cases would suggest a relation between the disease entity of chronic hepatitis without RBV or autoimmune markers and current or recent RBV infection.


Antibodies to hepatitis B core antigen in hepatitis B surface antigen-positive and -negative chronic hepatitis.

THE JOURNAL OF Il'\FECTIOUS DISEASES. VOL. 135, NO.6. JUNE 1977 © 1977 by the University of Chicago. All rights reserved. Antibodies. to Hepatitis B...
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