Vol. 13, No. 1 Printed in U.S.A.
INFECTION AND IMMUNITY, Jan. 1976, p. 296-297 Copyright ©) 1976 American Society for Microbiology
Correlation Between Fluorescent Antibody Detection of Hepatitis B Core Antigen in Liver Biopsies and the Presence of e Antigen in Serum B. L. MURPHY,* J. M. PETERSON,' J. L. SMITH, G. L. GITNICK,2 M. 0. AUSLANDER,2 K. R. BERQUIST, J. E. MAYNARD, AND R. H. PURCELL3 Phoenix Laboratories Division, Bureau ofEpidemiology, Center for Disease Control, Phoenix, Arizona 85014 Received for publication 6 August 1975
HB. Ag was detected by fluorescent antibody in liver biopsies taken from chronic hepatitis B surface antigen (HBS Ag) carriers having e antigen in their sera. HB(. Ag could not be detected in liver biopsies from similar individuals having e antibody in their sera.
Magnius and Espmark (7) in 1972 identified, by immunodiffusion procedures, a new and distinct antigen-antibody system in chronic carriers of hepatitis B surface antigen (HBS Ag) and designated the new antigenic determinant as "e." Unlike the previously described subspecificities of HB, Ag (a, d, y, w, r) (2, 6), the e determinant did not appear to be a surface component of hepatitis B particles. In 1974, Nielsen and colleagues (9) found an association between the presence of e antigen and persisting or chronic liver disease among HB, Ag carriers. Subsequently, Magnius and co-workers (8) reported that individual carriers of HB, Ag with antibody to e antigen appeared healthy and without evidence of liver dysfunction. They further hypothesized that e antigen was associated with the infectivity of HB, Ag-positive sera, perhaps through a relationship to the efficiency of Dane particle synthesis. Since the Dane particle probably represents the hepatitis B virus (HBV) itself (5, 10), carriers of HB, Ag and e antigen might be expected, under the hypothesis, to have larger quantities of circulating Dane particles than those carriers without e antigen. Recent description of the internal component of the Dane particle, now designated in part as hepatitis B core antigen (HB(. Ag) (1), together with published documentation of HB,. Ag synthesis in hepatocyte nuclei (4), provides further experimental basis for examining the hypothesis. Thus HB, Ag carriers of e antigen might be expected to demonstrate evidence of more effec' Present address: Medical Arts Associates, Ltd., Moline, DIl. 61265. 2 Present address: Department of Medicine, School of Medicine, UCLA Center for the Health Sciences, Los Angeles, Calif. 90024. 3Present address: Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md. 20014.
tive HB(. Ag synthesis than those carriers without e antigen. The following communication presents data bearing on this latter inference. Liver biopsies and/or necropsy liver specimens (chimpanzee and human) taken from long-term carriers of HB, Ag were examined by fluorescent antibody techniques for HB, Ag and HB,. Ag. All biopsies were of acceptable quality and allowed visualization and screening of at least 100 morphologically intact hepatocytes. Direct immunofluorescent staining of liver sections was performed using guinea pig hyperimmune anti-HB, and rhesus hyperimmune or human convalescent anti-HB(. labeled with fluorescein isothiocyanate by the methods of previously described test procedures (3). Positivity for HB, Ag by fluorescent antibody was demonstrated by perimembranous and/or cytoplasmic fluorescence. The finding of one or more fluorescent nuclei was considered positive for HB(. Ag staining. In addition, serum samples from these carriers were independently examined by rheophoresis (Abbott Laboratories) for e antigen and e antibody. The standard e antigen and e antibody reagents were derived from HB, Agpositive blood donor units and were kindly verified by George Le Bouvier, Yale University School of Medicine. The presence of HB, Ag in liver biopsy sections of chimpanzees and humans was positively correlated with the detection of e antigen, and similarly the absence of HB, Ag was correlated with e antibody in their sera (Table 1). Thus, HB, Ag was detected in biopsies of 10 subjects positive for e antigen and in only 1 of 6 subjects positive for e antibody. This difference is statistically significant (P < 0.002). The finding of HB, Ag in the liver of one chimpanzee with e antibody is of interest. On initial serological testing by rheophoresis, neither e antigen nor e antibody could be detected 296
NOTES
VOL. 13, 1976
TABLE 1. Results offluorescent antibody detection of
HB, Ag, HB, Ag, and the presence of e antigen or e antibody in serum e
antigen+
Total
Chimpanzees
5 5
HB, HB, Ag+ Ag+ 5
5
5 5
antibody+ Liver
Liver
Carriers
Humans ........
e
Total HB , HBc
Ag+ Ag+
2 4
2 3
1 0
in the serum. However, after further specific absorption tests, this serum was found to reduce the level of e activity in a standard e antigen reactant, thus suggesting the presence of a possible non-precipitating e antibody. For this reason the animal was included in the e antibody category. In one human HB5 Ag carrier with e antibody, HB5 Ag could not be demonstrated in liver biopsy section by fluorescent antibody, probably because of insufficient biopsy sample size. The above data support the inference that more active replication of HBV may be taking place in liver tissue of HB5 Ag carriers who are positive for e antigen than in those who are positive for e antibody. Since a single biopsy represents only a minute sample of the total mass ofhepatic tissue that might be involved in synthesis of HBV, some HBV replication may be taking place in carriers of HB5 Ag, regardless of e determinant status, even when HB, Ag is not detected in hepatocyte nuclei. Therefore, the consistent detection of HB, Ag in hepatocyte nuclei of e antigen-positive HB5 Ag carriers in contrast to the e antibody-positive carriers suggests that efficiency of HBV synthesis may be greater in the former instance. We have not had the opportunity to examine the sera from the subjects in this study by electron mi-
297
croscopy. However, a finding of an increased proportion of Dane particles relative to the other morphological forms of HB5 Ag in carriers with e antigen in comparison with e antibodypositive carriers would further support the hypothesis that the presence of e antigen is linked to HBV infectivity. We wish to acknowledge George Le Bouvier and Alan Williams, Yale University School of Medicine, for their advice and for performing the serum absorption tests.
1. 2.
3.
4.
5.
6.
7.
8.
9.
10.
LITERATURE CITED Almeida, J. D., D. Rubenstein, and E. J. Stott. 1971. New antigen-antibody system in Australia-antigenpositive hepatitis. Lancet 2:1225-1227. Bancroft, W. H., F. K. Mundon, and R. K. Russell. 1972. Detection of additional antigenic determinants of hepatitis B antigen. J. Immunol. 109:842-848. Berquist, K. R., J. M. Peterson, B. L. Murphy, J. W. Ebert, J. E. Maynard, and R. H. Purcell. 1975. Hepatitis B antigens in serum and liver of chimpanzees acutely infected with hepatitis B virus. Infect. Immun. 12:602-605. Edgington, T. S. 1974. Patterns of hepatitis B virus genome expression in vivo, p. 243-252. In W. S. Robinson and C. F. Fox (ed.), Mechanisms of virus disease. W. A. Benjamin Publishers, Inc., Menlo Park, Calif. Kaplan, P. M., R. L. Greenman, J. L. Gerin, R. H. Purcell, and W. S. Robinson. 1973. DNA polymerase associated with human hepatitis B antigen. J. Virol. 12:995-1005. Le Bouvier, G. L. 1971. The heterogeneity of Australia antigen. J. Infect. Dis. 123:671-675. Magnius, L. O., and J. A. Espmark. 1972. New specificities in Australia antigen-positive sera distinct from the Le Bouvier determinants. J. Immunol. 109:10171021. Magnius, L. O., A. Lindholm, P. Lundin, and S. Iwarson. 1975. A new antigen-antibody system: clinical significance in long term carriers of hepatitis B surface antigen. J. Am. Med. Assoc. 231:356-359. Nielsen, J. O., 0. Dietrichson, and E. Juhl. 1974. Incidence and meaning of the "e" determinant among hepatitis B antigen positive patients with acute and chronic liver diseases. Lancet 2:913-915. Robinson, W. S., D. A. Clayton, and R. L. Greenman. 1974. DNA of a human hepatitis B virus candidate. J. Virol. 14:384-391.
INFECTION AND IMMUNITY, Jan. 1976, p. 296-297 Copyright ©) 1976 American Society for Microbiology
Correlation Between Fluorescent Antibody Detection of Hepatitis B Core Antigen in Liver Biopsies and the Presence of e Antigen in Serum B. L. MURPHY,* J. M. PETERSON,' J. L. SMITH, G. L. GITNICK,2 M. 0. AUSLANDER,2 K. R. BERQUIST, J. E. MAYNARD, AND R. H. PURCELL3 Phoenix Laboratories Division, Bureau ofEpidemiology, Center for Disease Control, Phoenix, Arizona 85014 Received for publication 6 August 1975
HB. Ag was detected by fluorescent antibody in liver biopsies taken from chronic hepatitis B surface antigen (HBS Ag) carriers having e antigen in their sera. HB(. Ag could not be detected in liver biopsies from similar individuals having e antibody in their sera.
Magnius and Espmark (7) in 1972 identified, by immunodiffusion procedures, a new and distinct antigen-antibody system in chronic carriers of hepatitis B surface antigen (HBS Ag) and designated the new antigenic determinant as "e." Unlike the previously described subspecificities of HB, Ag (a, d, y, w, r) (2, 6), the e determinant did not appear to be a surface component of hepatitis B particles. In 1974, Nielsen and colleagues (9) found an association between the presence of e antigen and persisting or chronic liver disease among HB, Ag carriers. Subsequently, Magnius and co-workers (8) reported that individual carriers of HB, Ag with antibody to e antigen appeared healthy and without evidence of liver dysfunction. They further hypothesized that e antigen was associated with the infectivity of HB, Ag-positive sera, perhaps through a relationship to the efficiency of Dane particle synthesis. Since the Dane particle probably represents the hepatitis B virus (HBV) itself (5, 10), carriers of HB, Ag and e antigen might be expected, under the hypothesis, to have larger quantities of circulating Dane particles than those carriers without e antigen. Recent description of the internal component of the Dane particle, now designated in part as hepatitis B core antigen (HB(. Ag) (1), together with published documentation of HB,. Ag synthesis in hepatocyte nuclei (4), provides further experimental basis for examining the hypothesis. Thus HB, Ag carriers of e antigen might be expected to demonstrate evidence of more effec' Present address: Medical Arts Associates, Ltd., Moline, DIl. 61265. 2 Present address: Department of Medicine, School of Medicine, UCLA Center for the Health Sciences, Los Angeles, Calif. 90024. 3Present address: Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md. 20014.
tive HB(. Ag synthesis than those carriers without e antigen. The following communication presents data bearing on this latter inference. Liver biopsies and/or necropsy liver specimens (chimpanzee and human) taken from long-term carriers of HB, Ag were examined by fluorescent antibody techniques for HB, Ag and HB,. Ag. All biopsies were of acceptable quality and allowed visualization and screening of at least 100 morphologically intact hepatocytes. Direct immunofluorescent staining of liver sections was performed using guinea pig hyperimmune anti-HB, and rhesus hyperimmune or human convalescent anti-HB(. labeled with fluorescein isothiocyanate by the methods of previously described test procedures (3). Positivity for HB, Ag by fluorescent antibody was demonstrated by perimembranous and/or cytoplasmic fluorescence. The finding of one or more fluorescent nuclei was considered positive for HB(. Ag staining. In addition, serum samples from these carriers were independently examined by rheophoresis (Abbott Laboratories) for e antigen and e antibody. The standard e antigen and e antibody reagents were derived from HB, Agpositive blood donor units and were kindly verified by George Le Bouvier, Yale University School of Medicine. The presence of HB, Ag in liver biopsy sections of chimpanzees and humans was positively correlated with the detection of e antigen, and similarly the absence of HB, Ag was correlated with e antibody in their sera (Table 1). Thus, HB, Ag was detected in biopsies of 10 subjects positive for e antigen and in only 1 of 6 subjects positive for e antibody. This difference is statistically significant (P < 0.002). The finding of HB, Ag in the liver of one chimpanzee with e antibody is of interest. On initial serological testing by rheophoresis, neither e antigen nor e antibody could be detected 296
NOTES
VOL. 13, 1976
TABLE 1. Results offluorescent antibody detection of
HB, Ag, HB, Ag, and the presence of e antigen or e antibody in serum e
antigen+
Total
Chimpanzees
5 5
HB, HB, Ag+ Ag+ 5
5
5 5
antibody+ Liver
Liver
Carriers
Humans ........
e
Total HB , HBc
Ag+ Ag+
2 4
2 3
1 0
in the serum. However, after further specific absorption tests, this serum was found to reduce the level of e activity in a standard e antigen reactant, thus suggesting the presence of a possible non-precipitating e antibody. For this reason the animal was included in the e antibody category. In one human HB5 Ag carrier with e antibody, HB5 Ag could not be demonstrated in liver biopsy section by fluorescent antibody, probably because of insufficient biopsy sample size. The above data support the inference that more active replication of HBV may be taking place in liver tissue of HB5 Ag carriers who are positive for e antigen than in those who are positive for e antibody. Since a single biopsy represents only a minute sample of the total mass ofhepatic tissue that might be involved in synthesis of HBV, some HBV replication may be taking place in carriers of HB5 Ag, regardless of e determinant status, even when HB, Ag is not detected in hepatocyte nuclei. Therefore, the consistent detection of HB, Ag in hepatocyte nuclei of e antigen-positive HB5 Ag carriers in contrast to the e antibody-positive carriers suggests that efficiency of HBV synthesis may be greater in the former instance. We have not had the opportunity to examine the sera from the subjects in this study by electron mi-
297
croscopy. However, a finding of an increased proportion of Dane particles relative to the other morphological forms of HB5 Ag in carriers with e antigen in comparison with e antibodypositive carriers would further support the hypothesis that the presence of e antigen is linked to HBV infectivity. We wish to acknowledge George Le Bouvier and Alan Williams, Yale University School of Medicine, for their advice and for performing the serum absorption tests.
1. 2.
3.
4.
5.
6.
7.
8.
9.
10.
LITERATURE CITED Almeida, J. D., D. Rubenstein, and E. J. Stott. 1971. New antigen-antibody system in Australia-antigenpositive hepatitis. Lancet 2:1225-1227. Bancroft, W. H., F. K. Mundon, and R. K. Russell. 1972. Detection of additional antigenic determinants of hepatitis B antigen. J. Immunol. 109:842-848. Berquist, K. R., J. M. Peterson, B. L. Murphy, J. W. Ebert, J. E. Maynard, and R. H. Purcell. 1975. Hepatitis B antigens in serum and liver of chimpanzees acutely infected with hepatitis B virus. Infect. Immun. 12:602-605. Edgington, T. S. 1974. Patterns of hepatitis B virus genome expression in vivo, p. 243-252. In W. S. Robinson and C. F. Fox (ed.), Mechanisms of virus disease. W. A. Benjamin Publishers, Inc., Menlo Park, Calif. Kaplan, P. M., R. L. Greenman, J. L. Gerin, R. H. Purcell, and W. S. Robinson. 1973. DNA polymerase associated with human hepatitis B antigen. J. Virol. 12:995-1005. Le Bouvier, G. L. 1971. The heterogeneity of Australia antigen. J. Infect. Dis. 123:671-675. Magnius, L. O., and J. A. Espmark. 1972. New specificities in Australia antigen-positive sera distinct from the Le Bouvier determinants. J. Immunol. 109:10171021. Magnius, L. O., A. Lindholm, P. Lundin, and S. Iwarson. 1975. A new antigen-antibody system: clinical significance in long term carriers of hepatitis B surface antigen. J. Am. Med. Assoc. 231:356-359. Nielsen, J. O., 0. Dietrichson, and E. Juhl. 1974. Incidence and meaning of the "e" determinant among hepatitis B antigen positive patients with acute and chronic liver diseases. Lancet 2:913-915. Robinson, W. S., D. A. Clayton, and R. L. Greenman. 1974. DNA of a human hepatitis B virus candidate. J. Virol. 14:384-391.
