Journal of Clinical Pathology, 1978, 31, 681-687

Viral antigens and antibodies in hepatitis B infection C. R. HOWARD, A. R. ZANETTI,1 SARA THAL, AND A. J. ZUCKERMAN From the Department of Medical Microbiology and WHO Collaborating Centre for Reference and Research on Viral Hepatitis, London School of Hygiene and Tropical Medicine, Keppel Street, London WCJE 7HT, UK

Hepatitis B virus antigens and antibodies were detected in the sera of acute and persistently infected patients. Evidence of active virus replication was confined to immediately before or during the initial detection of hepatitis B surface antigen during acute hepatitis B. Hepatitis B core antibody appeared during the period of antigenaemia and preceded recovery. Hepatitis B e antigen was found in a proportion of sera which contained significant levels of virus particles. In contrast, all sera containing hepatitis B virus particles from persistently infected patients treated by maintenance haemodialysis also contained the e antigen. Among a group of 50 persistent carriers of hepatitis B virus, significant levels of virus production occurred in the presence of antibody to e antigen. In addition, evidence of exposure to hepatitis B virus was found among 3 % of blood donors in whose sera the surface antigen was not detected by radioimmunoassay. The significance of these findings is discussed in relation to the aetiology of hepatitis type B. SUMMARY

Sera containing hepatitis B surface antigen (HBsAg) Methods are now known to contain additional antigenic B SURFACE ANTIGEN AND specificities unrelated to the determinants present HEPATITIS ANTIBODY DETECTION on the surface antigen. The first of these is the core antigen, so termed because of its close association Commercially available solid phase radioimmunowith the inner component of the 42 nm particle assays were used for the detection of HBsAg and its described by Dane et al. (1970). This particle is antibody (Ausria II, Abbott Laboratories Inc, believed to represent the hepatitis B virus of man. North Chicago, Ill, USA). Where appropriate, the Core antigen may be detected after removal of the titre of HBsAg present was expressed as a ratio of outer surface antigen coat either by direct serological the sample counts per minute to the mean value examination or indirectly by assay of an integral obtained with 10 replicate samples of a confirmed serum. All assays were quantified DNA-dependent DNA polymerase activity (Kaplan HBsAg-negative et al., 1973). Magnius and Espmark (1972) recog- in an LKB Model 1280 gamma-counter to a standard nised a third precipitating antigen in some sera con- error of less than 4 %. taining the surface antigen, which they designated 'e'. DETECTION OF ANTIBODY TO CORE ANTIGEN The presence of the e antigen appears to correlate Antibody to hepatitis B core antigen (HBcAg) was with the replication of hepatitis B virus in the host carried out essentially as described by Howard and and, in general terms, with varying degrees of liver Zuckerman (1977). Trace labelled HBcAg was predamage (Nielsen etal., 1974). A significant correlation pared from a concentrated hepatitis B virus fraction between the presence of DNA polymerase activity, obtained from the serum of a chronically infected the presence of hepatitis B e antigen, and infectivity chimpanzee. Sera were tested at dilutions of 1/5 and has also been reported, although the significance of 1/50, and immune complexes were separated either the presence of antibody remains unclear. In the by binding to staphylococcal protein A or by the present study the presence of hepatitis B core anti- addition of donkey anti-human IgG (Wellcome gen, e antigen, and their respective antibodies was Reagents Ltd, Beckenham, Kent, UK). Samples that determined in individuals with acute or persistent precipitated more than 50 % of the added trace label infection with hepatitis B virus. were scored as positive. 'Present address: Instituto di Virologia dell' Universiti DETECTION OF E ANTIGEN AND ANTIBODY di Milano, Via Pascal, 38-20133, Milan, Italy. Hepatitis B e antigen (HBeAg) and its antibody were Received for publication 21 November 1977 681

C. R. Howard, A. R. Zanetti, Sara Thal, and A. J. Zuckerman

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detected either using the gel immunodiffusion method as described by Magnius and Espmark (1972) or by the use of gel rheophoresis plates (Abbott Laboratories Inc, North Chicago, Ill, USA). All positive samples were confirmed by their serological identity to reference reagents held at the WHO Collaborating Centre for Reference and Research on Viral Hepatitis. ELECTRON MICROSCOPY

Examination of sera and concentrated pellets of hepatitis B antigens was performed essentially as described by Krugman et al. (1974a). DETECTION OF CORE ANTIGEN-ASSOCIATED DNA POLYMERASE ACTIVITY

DNA polymerase activity in sera was quantified by a modification of the method originally described by Kaplan et al. (1973). Sera were concentrated by centrifugation overnight at 28 000 xg in an AR40.3 rotor or for four hours at 66 000 x g in an SW65 rotor. After resuspension into a 1/20th volume of PBS, Nonidet P40 and 2-mercaptoethanol were added to a final concentration of 07% and 0.2% respectively. The reaction mixture contained 16 ,umol tris pH 7.5, 4 ,umol MgCI2, 12 ,umol NH4CI, 5 nmol

each of dATP, dCTP, and dGTP, and approximately 35 pmol of 3H-thymidine methyl-5-triphosphate at 45-50 Ci/mmol (Radiochemical Centre, Amersham, Bucks, UK). After a two-hour incubation at 37'C two 50-,l volumes were spotted onto 3MM Whatman filter discs and dried at 60°C for 20 minutes, and each disc was individually treated with two 50-ml volumes of 50% trichloroacetic acid over 18 hours. Discs were then dehydrated in methanol, dried, and immersed in a toluene-based scintillation fluid for counting. Results DETECTION OF CORE ANTIGEN, E ANTIGEN, AND ANTIBODIES DURING ACUTE HEPATITIS B INFECTION

The appearance of core antigen-associated DNA polymerase activity as a marker of replication of the virus was investigated in a total of 109 sera collected serially at seven-day intervals from 43 patients with acute hepatitis B admitted to hospital. The amount of acid-insoluble product as a measure of DNA polymerase activity over a two-hour period of incubation is shown in Figure 1. Each result is expressed as a ratio of the counts per minute (cpm)

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Viral antigens and antibodies in hepatitis B infection

obtained with respect to that obtained with similarly treated negative control material. A ratio 2 or greater was regarded as being significant. The vertical bar on the right shows the range of values obtained with samples prepared in a similar manner from patients with acute viral hepatitis in whom HBsAg was not detected by radioimmunoassay. The highest values of DNA polymerase activity were recorded during the first two weeks of observation. The data obtained from two patients are shown in more detail in Figure 2. In patient (a) e antigen was still detectable in the absence of significant DNA polymerase activity. Its disappearance was closely followed by the detection of core antibody, but no antibody to the e antigen could be detected. In patient (b), although a similar rapid decline in the titre of the surface antigens was observed, e antibody was detected and appeared slightly ahead of the core antibody even though the latter was detected by a sensitive radioimmunoassay procedure. The appearance of e antibody suggests that in this case e antigen must have appeared much earlier in the infection before serum samples were available for examination. HEPATITIS B VIRAL ANTIGENS IN PATIENTS ON MAINTENANCE HAEMODIALYSIS

A correlation was sought between the core antigen associated DNA polymerase and e antigen in the sera of 16 patients undergoing maintenance haemodialysis for chronic renal failure. All had been persistent carriers of HBsAg for at least 12 months. A close correlation was found between significant levels of DNA polymerase activity and the presence of e antigen as detected by immunodiffusion (Fig. 3).

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The antigen was consistently found in the sera from 12 patients (75 %), 11 of which also contained high levels of DNA polymerase activity. In contrast, no significant incorporation was detected either in the presence of e antibody or in those samples in which e antigen or its antibody could not be found. In all cases a close correlation was also found between the level of DNA polymerase activity and the presence of virus particles as seen by electron microscopy. INCIDENCE OF HEPATITIS B VIRUS ANTIGEN IN HEALTHY BLOOD DONORS

A total of 50 asymptomatic carriers were examined for the presence of DNA polymerase, e antigen, and antibodies to hepatitis B virus antigens. The e antigen was found together with significant levels of DNA polymerase activity in only one instance (Fig. 4). However, in four carriers significant levels of enzyme activity were found in the presence of e antibody. Examination by electron microscopy of sera containing e antibody revealed intact virus particles in both the presence and absence of significant DNA polymerase levels (Fig. 5). In the remaining group of 16 sera, no evidence of e antigen or its antibody was found although five additional sera contained significant levels of DNA polymerase. Subsequent donations from several of these carriers suggested that there was some fluctuation in the level of DNA polymerase over periods of several months. During the course of these studies blood was regularly obtained from an asymptomatic chimpanzee carrier of hepatitis B virus for other studies. The results of DNA polymerase estimations on each sample are shown graphically in Figure 6.

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The level of activity varied by as much as a factor of four during a period of 14 months, cyclical variations occurring every four to six months. The presence of hepatitis B core antibody was also found in all but one of the 50 human asymptomatic carriers (Table). In contrast, only 63 % of sera from

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with replication of the virus and with infectivity (Alter et al., 1976). Krugman et al. (1974b) examined serial serum samples from volunteers who had been inoculated with the MS-2 strain of hepatitis B virus. Hepatitis B surface antigen was detected four weeks after parenteral inoculation and was followed 10 days later by a rise in serum DNA polymerase Both events preceded elevation of the serum activity. Table Detection of antibody during acute and chronic transaminase levels. In a similar study of three hepatitis B infection patients who received multiple transfusions, Kaplan No. tested anti-HBs anti-HBc et al. (1974) found DNA polymerase activity in the positive positive serum approximately seven days after the appearance of hepatitis B surface antigen but it disappeared HBsAg positive Acute hepatitis sera 35 1 (3 %) 26 (74%) before the peak of transaminase elevation was 11 0 9 Haemodialysis patients reached. In the present study, significant levels of 5 (10%) 49 (98%) Asymptomatic chronic carriers 50 DNA polymerase activity were found in 31 of 109 HBsAg negative sera obtained from cases of acute hepatitis B. The 68 68 (100%) 43 (63 %) Late convalescent sera 97 1 (1 %) 3 (3 %) Healthy blood donors majority of positive findings were confined to the 2 0 Hyperimmune sera to HBsAg 2 first two weeks of observation (Fig. 1). In a recent 2 0 Immunoglobulin preparations 2 study, Cappel et al. (1977) also found the appearance of DNA polymerase activity to be transient, signi68 healthy blood donors with surface antibody con- ficant levels of enzyme being detected five days before tained also core antibody. Surprisingly, another 3 % the onset of clinical hepatitis in persons who became of donors without detectable surface antigen and infected in a high-risk setting. No attempt was made, antibody were found to have hepatitis B core anti- however, to monitor the presence of either e antigen body. No antibody to core antigen could be found or its antibody. in two preparations of immunoglobulin containing Among the acute hepatitis sera examined in our high levels of surface antibody together with two study, only three were found to contain both e hyperimmune sera prepared in laboratory animals antigen and DNA polymerase activity. Antibody to to purified surface antigen which were included as e antigen was detected in one instance. The antigen controls. was also detected in three other sera lacking significant DNA polymerase activity, whereas e antibody Discussion was present in six sera, although generally confined to the last sample obtained from the patient. In conThe finding of a DNA polymerase activity within the trast, core antibody was detected as early as the 42 nm virus particles permitted the development of second week in all of 16 patients tested. In several a unique assay for the presence of the virus. The instances, the combined serological findings revealed enzyme is closely associated with an essentially that an e antibody response may precede the double-stranded circular DNA template (Robinson, appearance of core antibody (Fig. 2). It is noteworthy 1974), and its presence in serum has been associated that e antibody was detected in earlier samples using

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C. R. Howard, A. R. Zanetti, Sara Thal, and A. J. Zuckerman

relatively insensitive techniques of immunodiffusion whereas core antibody was detected by a radioimmunoassay procedure (Howard and Zuckerman, 1977). In one of the two cases illustrated, e antigen was clearly detected in the absence of DNA polymerase activity. In contrast, a strong correlation between the appearance of e antigen and circulating DNA polymerase activity was found in 11 persistently infected patients undergoing maintenance haemodialysis. Sera from all these patients contained the 42 nm virus particles when examined by electron microscopy in contrast to two other patients with antibody in the absence of DNA polymerase activity. Nordenfelt and Kjellen (1975) also found a similar correlation between e antigen, DNA polymerase activity, and viral replication in a small number of patients undergoing maintenance haemodialysis. Nielsen et al. (1974) reported that hepatitis B e antigen was significantly more common in patients with chronic hepatitis and cirrhosis and hepatitis B surface antigen than in patients with acute viral hepatitis and suggested that the presence of the e antigen may be a valuable prognostic marker of progression to chronic liver disease. However, the possibility that the appearance of the e antigen is a host response has yet to be definitely excluded (El Sheikh et al., 1975; Perrillo et al., 1977). Several investigations have been directed to the relationship between the e antigen and replication of hepatitis B virus in asymptomatic persistent carriers since Magnius and Espmark originally suggested that a proportion of blood donors with e antigen may carry a greater risk of transmitting hepatitis B infection. In a study of over 400 such blood donors in Japan, Imai et al. (1976) found that 14% had circulating hepatitis B e antigen and a further 24% possessed e antibody. Findings of specific DNA polymerase activity were confined to sera with e antigen, and a further 15 % of the remaining samples were found to be free of both antigen and antibody. In the present study a total of 50 persistent carriers were similarly tested. The sera from 10 of these (20%) contained significant levels of DNA polymerase activity, but e antigen was found concurrently in only one carrier. A further four were found to have e antibody in the presence of the enzyme, and electron microscopy confirmed the presence of hepatitis B virus particles in two of these. Neither e antigen nor its antibody could be detected in the remaining samples with significant DNA polymerase activity. Resolution of this group requires the development of more sensitive techniques for the detection of e antigen and its antibody. Liver biopsies were not available in order to assess the extent, if any, of liver damage in either category. Feinman et

al. (1975), however, reported an association of e antibody with a normal or slightly abnormal liver, but they did not relate this to the presence of circulating HBV. In agreement with previous reports (Tsuda et al., 1975; Hoofnagle et al., 1977), almost all of the persistent carriers investigated possessed hepatitis B core antibody. In the present study a group of 97 healthy volunteer blood donors in whose serum the surface antigen was not detected by radioimmunoassay were also examined. Of these, 3 % were found to contain core antibody in the absence of all other indicators of infection with hepatitis B virus. Other studies have also indicated the possibility of recent or ongoing infection, hepatitis B taking place in certain individuals in the absence of detectable surface antigen. Tsuda et al. (1975) reported that 3 % of blood donors in Japan had core antibody as detected by an immune adherence haemagglutination method in the absence of surface antigen and antibody. Gerber et al. (1977) found core antibody in 21 % of 33 patients with surface antigen-negative chronic hepatitis, and Irwin et al. (1977) have recently reported that 39 % of surface antigennegative acute hepatitis patients in a military population had core antibody. It would therefore seem likely that active replication of this virus may occur in some patients in the absence of excess production of the surface antigen in the form of 20 to 25 nm particles. The lack of excess surface antigen-bearing material may lead to the failure of sensitive radioimmunoassay procedures to detect material that may contain only circulating complete virus particles. It has been suggested that infection with hepatitis B virus may proceed in a manner that results in a large number of defective particles necessary for the establishment and subsequent maintenance of virus persistence (Gerin et al., 1975). This hypothesis is strengthened by the finding of apparently empty 42 nm virus particles and the fluctuations in DNA polymerase activity observed over a period of time in the one persistently infected chimpanzee. This may represent an underlying mechanism of defective particle production that is not dissimilar to the proposed mechanism of vesicular stomatitis virus persistent infection in tissue culture (Sinarachatanant and Huang, 1977). The study of antigens and antibodies associated with hepatitis B virus in acutely and persistently infected individuals is of immense value in the understanding of pathogenesis of this infection. The association of e antigen with infectivity and the possible significance of the production of core antibody in exposed persons will be essential factors in the evaluation of the experimental hepatitis B vaccines currently under development.

Viral antigens and antibodies in hepatitis B infection We are indebted to Miss Jill Preece for excellent technical assistance. The hepatitis research programme is supported by the World Health Organisation, the Medical Research Council, the Wellcome Trust, and the Department of Health and Social Security. One of us (ARZ) was in receipt of a CNR award during the course of this work. References Alter, H. J., Seeff, L. B., Kaplan, P. M., McAuliffe, V. J., Wright, E. C., Gerin, J. L., Purcell, R. H., Holland, P. V., and Zimmerman, H. J. (1976). Type B hepatitis: the infectivity of blood positive for e antigen and DNA polymerase after accidental needlestick exposure. New England Journal of Medicine, 295, 909-913. Cappel, R., de Cuyper, F., van Beers, D., Toppet, M., and Cadranel, S. (1977). Early diagnosis of hepatitis B by Dane particle associated DNA polymerase assay. Journal of General Virology, 36, 217-220. Dane, D. S., Cameron, C. H., and Briggs, M. (1970). Virus-like particles in serum of patients with Australiaantigen-associated hepatitis. Lancet, 1, 695-698. El Sheikh, N., Woolf, I. L., Galbraith, R. M., Eddleston, A. L. W. F., Dymock, I. W., and Williams, R. (1975). e Antigen-antibody system as indicator of liver damage in patients with hepatitis-B antigen. British Medical Journal, 4, 252-253. Feinman, S. V., Berris, B., Sinclair, J. C., Wrobel, D. M., Murphy, B. L., and Maynard, J. E. (1975). e Antigen and anti-e in HBsAg carriers. Lancet, 2, 1173-1174. Gerber, M. A., Zappi, T., Vernace, S. J., and Paronetto, F. (1977). Antibodies to hepatitis B core antigen in hepatitis B surface antigen-positive and -negative chronic hepatitis. Journal of Infectious Diseases, 135, 1006-1009. Gerin, J. L., Ford, E. C., and Purcell, R. H. (1975). Biochemical characterization of Australia antigen. American Journal ofPathology, 81, 651-661. Hoofnagle, J. H., Gerety, R. J., and Barker, L. F. (1973). Antibody to hepatitis-B-virus core in man. Lancet, 2, 869-873. Howard, C. R., and Zuckerman, A. J. (1977). Core antigen and circulating anti-core antibody in hepatitis B infection. Journal of Immunological Methods, 14, 291-301. Imai, M., Tachibana, F. C., Moritsugu, Y., Miyakawa, Y., and Mayumi, M. (1976). Hepatitis B antigenassociated deoxyribonucleic acid polymerase activity and e Antigen/Anti-e system. Infection and Immunity, 14, 631-635. Irwin, G. R., Allen, R. G., Segal, H. G., Allen, A. M., Putnak, J. R., Cannon, H. G., and Top, F. H., Jr.

687 (1977). Serodiagnosis of hepatitis B virus infection by antibody to core antigen. Journal of Infectious Diseases, 136, 31-36. Kaplan, P. M., Gerin, J. L., and Alter, H. J. (1974). Hepatitis B-specific DNA polymerase activity during post-transfusion hepatitis. Nature, 249, 762-763. Kaplan, P. M., Greenman, R. L., Gerin, J. L., Purcell, R. H., and Robinson, W. S. (1973). DNA polymerase associated with human hepatitis B antigen. Journal of Virology, 12, 995-1005. Krugman, S., Bird, R. G., and Zuckerman, A. J. (1974a). Characterization of MS-2 (hepatitis B) serum by electron microscopy. Journal of Infectious Diseases, 130, 416-418. Krugman, S., Hoofnagle, J. H., Gerety, R. J., Kaplan, P. M., and Gerin, J. L. (1974b). Viral hepatitis, type B: DNA polymerase activity and antibody to hepatitis B core antigen. New England Journal of Medicine, 290, 1331-1335. Magnius, L. 0., and Espmark, J. A. (1972). New specificities in Australia antigen positive sera distinct from the Le Bouvier determinants. Journal of Immunology, 109, 1017-1021. Nielsen, J. 0., Dietrichson, 0., and Juhl, E. (1974). Incidence and meaning of the 'e' determinant among hepatitis-B-antigen positive patients with acute and chronic liver diseases. Lancet, 2, 913-915. Nordenfelt, E., and Kjell6n, L. (1975). Dane particles, DNA polymerase, and e-antigen in two different categories of hepatitis B antigen carriers. Intervirology, 5, 225-232. Perrillo, R. P., Gelb, L. D., Milligan, W. H., III, Wellinghoff, W., and Aach, R. D. (1977). Discordant e antigen, DNA polymerase activity, and Dane particle responses in two patients representing an index case-contact case pair with hepatitis B virus infection. Journal of Infectious Diseases, 136, 117-121. Robinson, W. S. (1974). A human hepatitis B virus candidate. In Mechanisms of Virus Disease, edited by W. S. Robinson and C. F. Fox, pp. 225-241. W. A. Benjamin, Menlo Park, California, USA. Sinarachatanant, P., and Huang, A. S. (1977). Effects of temperature and antibody on the cyclic growth of vesicular stomatitis virus. Journal of Virology, 21, 161-167. Tsuda, F., Takahashi, T., Takahashi, K., Miyakawa, Y., and Mayumi, M. (1975). Determination of antibody to hepatitis B core antigen by means of immune adherence hemagglutination. Journal of Immunology, 115, 834-838. Requests for reprints to: Professor A. J. Zuckerman, Department of Medical Microbiology, London School of Hygiene and Tropical Medicine, Keppel Street, Gower Street, London WC1E 7HT.

Viral antigens and antibodies in hepatitis B infection.

Journal of Clinical Pathology, 1978, 31, 681-687 Viral antigens and antibodies in hepatitis B infection C. R. HOWARD, A. R. ZANETTI,1 SARA THAL, AND...
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