hepatitis b e antigen and antibody measured by radioimmunoassay in acute hepatitis b surface...

Hepatitis B e Antigen and Antibody Measured by Radioimmunoassay in Acute Hepatitis BSurface Antigen-Positive HepatitisAuthor(s): J. Aldershvile, G. G. Frösner, J. O. Nielsen, F. Hardt, F. Deinhardt, P. Skinhøj andCopenhagen Hepatitis Acuta ProgrammeSource: The Journal of Infectious Diseases, Vol. 141, No. 3 (Mar., 1980), pp. 293-298Published by: Oxford University PressStable URL: http://www.jstor.org/stable/30081699 .

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THE JOURNAL OF INFECTIOUS DISEASES * VOL. 141, NO. 3 * MARCH 1980 @ 1980 by The University of Chicago. 0022-1899/80/4103-0004$00.75

Hepatitis B e Antigen and Antibody Measured by Radioimmunoassay in Acute Hepatitis B Surface Antigen-Positive Hepatitis

J. Aldershvile, G. G. Frosner, J. O. Nielsen, F. Hardt, F. Deinhardt, P. Skinh0j, and the

Copenhagen Hepatitis Acuta Programme

From the Medical Department II, Kommunehospitalet; the Division of Hepatology and Department of Pathology,

Hvidovre Hospital; and the Department of Infectious Diseases M, Rigshospitalet, Copenhagen, Denmark; and the

Max von Pettenkofer-Institut, University of Munich, Federal Republic of Germany

The presence of the hepatitis B e antigen (HBeAg) and antibody to HBeAg (anti-HBe) was studied by a solid-phase radioimmunoassay in 70 patients with acute hepatitis positive for hepatitis B surface antigen. HBeAg was found in 15 of 16 patients studied within two weeks after onset of symptoms. In total, 45 patients were found positive for HBeAg. The prevalence of this antigen declined constantly during the first 10 weeks after onset of symptoms. Chronic liver disease was demonstrated or suspected in 10 patients, including the five patients who had HBeAg for more than 10 weeks after onset of symptoms. In 33 patients with seroconversion from HBeAg to anti-HBe, 27 (82%) developed anti-HBe within two weeks after the clearance of HBeAg. Among the 58 patients who were or became HBeAg-negative, 53 (91 %) had or developed anti-HBe. Thus HBeAg seems to be present regularly in early acute hepatitis B. The persistence of HBeAg may be of prognostic value for the development of a chronic carrier state with hepatitis B surface antigen and/or a chronic liver disease.

In 1972 Magnius and Espmark described [1, 2] a new group of precipitating antigens-the e-antigen (HBeAg)-related to hepatitis B virus infections. In later studies the HBeAg was found to be closely related to the presence of circulating Dane particles [3-5] and core-specific DNA polymerase activity [4-6] in serum. In agreement with these findings, other investigators also found that the HBeAg seemed to be a marker for infectivity [7-9].

In 1974 we reported [3] results that indicated a correlation between HBeAg in acute hepatitis B and later development of chronic liver disease.

Received for publication July 20, 1979, and in revised form October 9, 1979.

Other members of the Copenhagen Hepatitis Acuta Pro-

gramme were: P. Christoffersen, O. Dietrichson, P. Elling, V.

Faber, C. Gluud, G. Haybye, K. Iversen, E. Juhl, A. F. Jor- gensen, L. Mathiesen, S. Norby-Rasmussen, P. Petersen, H.

Poulsen, K. Rams0e, P. Schlichting, K. Sloth, T. I. A. Soren- sen, and U. Tage-Jensen.

This study was supported by the Danish Medical Research Council (grants no. 512-10225, no. 512-10136, and no. 512-

8202), K0benhavns Kommunehospitals 100 ars jubilaemsfond, Ebba Celinders Foundation, Kobmand i Odense Johann og Hanne Weimann f. Seedorffs Foundation, and the Deutschen

Forschungsgemeinschaft (Fr. 400/6). Please address requests for reprints to Dr. J. Aldershvile,

Division of Hepatology 233, University of Copenhagen, Hvi- dovre Hospital, DK 2650 Hvidovre, Denmark.

These results have been confirmed by some groups [10-12], while others have found no such correlation [13, 14]. The discrepancy between these studies may be due to the drawing of sera at different times during the acute phase of the disease and the different methods for determination of HBeAg. The presence of antibody to HBeAg (anti-HBe) has in several studies been correlated with the "healthy" carrier state for hepatitis B surface antigen (HBsAg) [15, 16] and with low or absent infectivity of serum [9, 17].

In 1978 Frosner et al. as well as Mushahwar et al. described a highly sensitive solid-phase radioimmunoassay (RIA) for detection of HBeAg and anti-HBe [18, 19].

The purpose of the present study is to elucidate the incidence and meaning of HBeAg and anti- HBe during the course of acute type B hepatitis, as measured by RIA.

Materials and Methods

Patients. In the period from January 1, 1971, to May 1, 1972, 200 patients with biopsy-verified acute hepatitis entered the "Copenhagen Hepatitis Acuta Programme" [3]. Seventy of these patients had circulating HBsAg in the first available serum sample and were included in the study.

293



294 Aldershvile et al.

During the period of hospitalization (mean, 4.5 weeks) serum samples were collected weekly. Fur- ther samples were taken at the frequent follow-up examinations. The mean age of the patients was 23 years (range, 15-80 years); 48 were men, and 40 were drug addicts (29 males).

All patients were questioned for the interval between occurrence of first symptoms and admittance to the hospital. The following symptoms were accepted as being correlated with onset of hepatitis: jaundice, dark urine, acholic feces, up- per gastrointestinal complaints, joint complaints, and fever.

HBeAg and anti-HBe. For the RIA, an IgG preparation of human serum positive for anti-HBe (M.W.W.) was labeled with [1251] as previously described [19-21]. HBeAg was determined as previously described [21]. Beads coated with anti- HBe were incubated twice overnight with the test serum and with labeled anti-HBe. All serum samples that showed a binding of radioactivity higher than 2.1 times that of the negative control were considered positive for HBeAg. To test the speci- ficity of the HBeAg-positive samples, we determined anti-HBe by a blocking test with use of two overnight incubations of HBeAg-coated beads with test serum and with labeled anti-HBe. This method has been described in detail elsewhere [21]. Serum samples that exhibited binding of radioactivity >50% than that of the negative control were considered false-positive for HBeAg. False- positives were found with some serum samples positive for rheumatoid factors.

Anti-HBe. For the anti-HBe determination, a neutralization test was used. In this assay 100 pl of a predetermined dilution of a HBeAg-positive serum (H.M.) was incubated overnight with 100 pl of the serum to be tested. Then an anti-HBe-coated bead was added to the mixture. After a second overnight incubation the bead was washed and incubated again overnight with a 125I-labeled anti- HBe-IgG preparation. After washing, the bound radioactivity was counted. All serum samples that neutralized >50%0 of the predetermined quantity of HBeAg were considered positive for anti-HBe.

In addition, all sera were tested for HBeAg and anti-HBe by immunodiffusion as previously described [3].

HBsAg and antibody to HBsAg (anti-HBs). HBsAg and anti-HBs were demonstrated by immunodiffusion [3], and all sera negative by this

method were further tested by a solid-phase RIA (Ausria@ II-125 and AusabR, Abbott Laborato- ries, North Chicago, Ill.).

Results

All 70 patients who entered the study had circulating HBsAg at time of entrance. Three of these patients showed nonspecific HBeAg reactions and could therefore not be classified according to the HBe markers. These three patients are included only in table 1.

Table 1 shows the serologic findings in the first available sera from 70 patients. Among the 45 patients who had HBeAg, 32 developed anti-HBe. In four of these 45 patients no anti-HBe was demonstrated during the time of observation. The remaining nine patients were HBeAg-positive in the last available serum sample. Four of these nine patients were followed for <10 weeks, one was followed for 18 weeks, and the remaining four patients were followed for a period between three and six years. Nine patients had no HBe markers at the time of admission. Eight of these subse- quently developed anti-HBe, and one of these had transient HBeAg before the development of anti- HBe. Of the 67 patients in whom HBeAg/anti- HBe determination could be performed, only one was without HBe markers during the observation period of 2.5 years. All 13 patients who had anti- HBe on arrival at the hospital remained positive for anti-HBe during the entire follow-up period.

Table 1. Results of serologic tests in 70 patients with acute viral hepatitis who were positive for hepatitis B surface antigen at the time of admission to the hospital.

No. (%) with specific result

Radio- Immuno- Results* immunoassay diffusion

HBeAg-positive anti-HBe-negative 43 (61) 4 (6)

HBeAg-positive anti-HBe-positive 2 (3) 0

HBeAg-negative anti-HBe-positive 13 (19) 0

No HBe markers 9 (13) 66 (94) Nonspecific HBeAg

reactions 3 (4) 0

Total 70 (100) 70 (100)

* HBeAg = hepatitis B e antigen; anti-HBe = antibody to HBeAg.



HBeAg and Anti-HBe in Acute Hepatitis B 295

In contrast to the results obtained with RIA, immunodiffusion detected HBeAg in only four patients and anti-HBe in none (table 1).

In table 2 the serologic findings (RIA) in the first serum samples are given in relation to onset of symptoms in the 60 patients in whom time of first symptom could be established. All except one patient were HBeAg-positive when tested within the first two weeks after onset of symptoms. Among patients admitted to the hospital at a later stage of the disease, fewer were HBeAg-positive. Parallel to this the first anti-HBe-positive patients were detected three weeks after onset of first symptoms, and the frequency of anti-HBe-positive patients rose during the following weeks.

Figure 1 shows that the number of patients remaining HBeAg-positive declined constantly during the first 10 weeks after onset of symptoms. Five of the 45 patients who were HBeAg-positive at the time of admission to the hospital remained HBeAg-positive for >10 weeks after onset of symptoms. A chronic HBsAg carrier state (HBsAg- positive for more than six months) was docu- mented in four of the five patients. When the fifth patient was lost to follow-up after 18 weeks, HBsAg and HBeAg were still present. All four HBsAg carriers remained positive for HBsAg and HBeAg during the entire period of observation (three to six years). Thirty-six patients cleared the HBeAg within 10 weeks after their first symptoms, and none of these patients developed a HBsAg carrier state. The remaining four patients were lost to follow-up studies before 10 weeks after onset of symptoms. Thus the likelihood of a patient being a HBsAg carrier seemed closely correlated to the presence of HBeAg for >10 weeks after onset of symptoms.

The patient who was lost to follow-up after 18 weeks still had biochemical findings compatible

with active liver disease. The follow-up biopsies in the four chronic HBsAg carriers revealed histo- logic changes compatible with chronic persistent hepatitis in one, portal fibrosis in one, confluent necrosis in one, and cirrhosis in one. The follow- up biopsies of the remaining patients revealed chronic persistent hepatitis in another four patients and confluent necrosis in one. None of these had persistent HBeAg or HBsAg. On admittance to the hospital one of these five patients had both HBeAg and anti-HBe; one was HBeAg-positive for seven weeks but did not develop anti-HBe within the following six years, and one had anti- HBe. One patient who was HBeAg-positive for four weeks developed anti-HBe more than six weeks after the clearance of HBeAg, and one developed anti-HBe more than six weeks after admittance, but never had detectable HBeAg. Thus in our study, all the five patients who were HBeAg- positive for >10 weeks after onset of symptoms developed signs of chronic liver disease, in comparison with five of the 58 patients who either had anti-HBe or cleared the HBeAg within 10 weeks after their first symptoms.

Of the total of 45 HBeAg-positive patients, 33 had a seroconversion from HBeAg to anti-HBe. For these 33 patients the cumulative number of patients developing anti-HBe is given in correlation to number of weeks passed since the clearing of HBeAg in figure 2. Two patients simultaneous- ly had HBeAg and anti-HBe in one serum sample. Of the 33 patients, 27 (82%) had detectable anti- HBe two weeks after clearing of HBeAg. Three of the patients developed anti-HBe more than six weeks after disappearance of HBeAg.

Discussion

In a previous report [3] we suggested that HBeAg

Table 2. Serologic results by immunoassay in the initial serum samples in relation to the time in weeks after onset of first symptoms in 60 patients with acute type B hepatitis.

No. (%) with result at indicated week(s) after onset of first symptom

Results* 1 2 3 4 5 6 7 10

HBeAg-positive, anti-HBe-negative 5 (83) 10 (100) 8 (56) 8 (47) 4 (57) 2 (50) 1 HBeAg-positive, anti-HBe-positive 2 (12) No HBe markers 1 (17) 3 (22) 4 (23) Anti-HBe-positive 3 (22) 3 (18) 3 (43) 2 (50) 1

Total 6 10 14 17 7 4 1 1 * HBeAg = hepatitis B e antigen; anti-HBe = antibody to HBeAg.



296 Aldershvile etal.

Number of HBeAg positive patients

(100o/) 45

(89 %/) 40

(78%) 35

(67 %) 30

(56 %) 25

(44 %) 20

(330/1) 15

(22%) 10

(11%) 5

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 17 Weeks

Figure 1. The duration of antigenemia with the hepatitis B e antigen (HBeAg) by radioimmunoassay in the 45 patients who were HBeAg-positive on admission to the hospital as indicated by the number of patients still HBeAg- positive at each week after onset of symptoms; = 40 HBeAg-positive patients with established first symptom; I = five HBeAg-positive patients without known first symptom (time is given in weeks after admission); T = patients who were lost to follow-up before clearance of HBeAg; e = patients positive for HBeAg by immunodiffusion.

might be a valuable prognostic marker in predict- ing chronicity. This has been supported by some investigators [10-12], but others [13, 14] have questioned such a strong correlation. However, it has been difficult to compare these studies because the sera tested by the different groups have probably not been drawn at the same stage of the acute hepatitis. Furthermore, the assays applied had a lower and variable sensitivity.

Our results with RIA suggest that the presence of HBeAg for >10 weeks after first symptom pre- dicts a chronic HBsAg/HBeAg carrier state with high risk of development of chronic liver disease. This suggestion is in accordance with the findings by Norkrans et al. [22].

In the present study using RIA determinations, we could find HBe markers in 66 of the 70 pa-

tients, in comparison with only four of 70 when immunodiffusion tests were used.

In immunodiffusion tests HBeAg has been an inconsistent finding in the acute phase of hepatitis B. With use of solid-phase RIA, 15 of 16 patients were found to be TReAg-positive when tested within two weeks after onset of the disease. This indicates clearly that HBeAg is a constant phe- nomenon in acute type B hepatitis, a finding parallel to that of previous studies [21, 23].

The solid-phase RIA applied in this study has proved to be at least 500 times more sensitive than immunodiffusion [19, 21]. Sera showing an ele- vated cpm value (as compared to the negative control) in the anti-HBe blocking test were considered nonspecifically positive [21]. Fewer than 2% of the serum samples tested showed this reaction.



HBeAg andAnti-HBe in Acute Hepatitis B 297

Figure 2. Cumulative curve of appearance of antibody (anti-HBe) to hepatitis B e antigen (HBeAg) in 33 patients with acute type B hepatitis, who had a seroconversion from HBeAg to anti-HBe.

The accumulated number of patients with anti-HBe

(100%) 33 -

(91%) 30-

(76%) 25-

(61%) 20

(45"%) 15

(30%) 10

(15%) 5

0 1 2 3 4 5 6 >6 Weeks passed since

clearing of HBe Ag

The anti-HBe neutralization test used in this study demonstrated that nearly all patients with acute type B hepatitis show a seroconversion with early development of anti-HBe. This finding was in contrast to the findings by Norkrans et al. [22], who, however, used a blocking anti-HBe assay. In our laboratory the neutralization assay has been twice as sensitive as the blocking assay (authors' unpublished observations), a difference that might explain these different findings.

Early appearance (earlier than two weeks) of anti-HBe after the clearing of the HBeAg may in- dicate an uncomplicated course of the acute hepatitis, whereas a delayed appearance (later than six weeks) might predict a prolonged course, including the development of chronic persistent hepatitis.

References

1. Magnius, L. O., Espmark, J. A. New specificities in Aus- tralia antigen positive sera distinct from the Le Bouvier determinants. J. Immunol. 109:1017-1021, 1972.

2. Magnius, L. O., Espmark, J. A. A new antigen complex co-occurring with Australia antigen. Acta Pathol. Mi- crobiol. Scand. [B] 80:335-337, 1972.

3. Nielsen, J. O., Dietrichson, O., Juhl, E. Incidence and meaning of the "e" determinant among hepatitis-B- antigen positive patients with acute and chronic liver diseases. Lancet 2:913-915, 1974.

4. Hindman, S. H., Gravelle, C. R., Murphy, B. L., Bradley, D. W., Budge, W. R., Maynard, J. E. "e" antigen, Dane particles, and serum DNA polymerase activity in HBsAg carriers. Ann. Intern. Med. 85:458-460, 1976.

5. Nordenfelt, E., Andren-Sandberg, M. Dane particle-asso-

ciated DNA polymerase and e antigen: relation to chronic hepatitis among carriers of hepatitis B surface antigen. J. Infect. Dis. 134:85-89, 1976.

6. Cappel, R., De Cuyper, F., van Beers, D. e antigen and antibody, DNA polymerase, and inhibitors of DNA polymerase in acute and chronic hepatitis. J. Infect. Dis. 136:617-622, 1977.

7. Alter, H. J., Seeff, L. B., Kaplan, P. M., McAuliffe, V. J., Wright, E. C., Gerin, J. L., Purcell, R. H., Hol- land, P. V., Zimmerman, H. J. Type B hepatitis: the infectivity of blood positive for e antigen and DNA polymerase after accidental needlestick exposure. N. Engl. J. Med. 295:909-913, 1976.

8. Grady, G. F. Report of the U.S. National Heart and Lung Institute Collaborative Study Group and Phoenix Labo- ratories Division, Bureau of Epidemiology, Center for Disease Control. Relation of e antigen to infectivity of HBsAg-positive inoculations among medical personnel. Lancet 2:492-493, 1976.

9. Okada, K., Kamiyama, I., Inomata, M., Imai, M., Miya- kawa, Y., Mayumi, M. e antigen and anti-e in the serum of asymptomatic carrier mothers as indicators of positive and negative transmission of hepatitis B virus to their infants. N. Engl. J. Med. 294:746-749, 1976.

10. Tabor, E., Gerety, R. J., Barker, L. F. Detection of e antigen during acute and chronic hepatitis B virus infections in chimpanzees. J. Infect. Dis. 136:541-547, 1977.

11. Eleftheriou, N., Thomas, H. C., Heathcote, J., Sherlock, S. Incidence and clinical significance of e antigen and antibody in acute and chronic liver disease. Lancet 2: 1171-1173, 1975.

12. Norkrans, G., Magnius, L., Iwarson, S. e antigen in acute hepatitis B. Br. Med. J. 1:740-742, 1976.

13. Fay, O., Tanno, H., Roncoroni, M., Edwards, V. M., Mosley, J. W., Redeker, A. G. Prognostic implications of the e antigen of hepatitis B virus. J.A.M.A. 238: 2501-2503, 1977.

14. Gibson, P. E., Ruparelia, K. Occurrence of e antigen in acute hepatitis B. J. Clin. Pathol. 30:925-927, 1977.



298 Aldershvile et al.

15. Trepo, C. G., Magnius, L. O., Schaefer, R. A., Prince, A. M. Detection of e antigen and antibody: correlations with hepatitis B surface and hepatitis B core antigens, liver disease, and outcome in hepatitis B infections. Gas- troenterology 71:804-808, 1976.

16. Magnius, L. O., Lindholm, A., Lundin, P., Iwarson, S. A new antigen-antibody system. Clinical significance in long-term carriers of hepatitis B surface antigen. J.A.M.A. 231:356-359, 1975.

17. Skinhoj, P., Cohn, J., Bradburne, A. F. Transmission of hepatitis type B from healthy HBsAg-positive mothers. Br. Med. J. 1:10-11, 1976.

18. FrOsner, G. G., Sugg, U., Haas, H., Overby, L. R., Dein- hardt, F. Solid-phase radioimmunoassay for HBeAg and anti-HBe. Fresenius Zeitschrift fuer Analytische Chemie 290:185, 1978.

19. Mushahwar, I. K., Overby, L. R., Fr6sner, G., Deinhardt, F., Ling, C.-M. Prevalence of hepatitis B e antigen and its antibody as detected by radioimmunoassay. J. Med. Virol. 2:77-87, 1978.

20. Greenwood, F. C., Hunter, W. M., Glover, J. S. The preparation of ['3'I]-labeled human growth hormone of high specific radioactivity. J. Biochem. 89:114-123, 1963.

21. Frosner, G. G., Brodersen, M., Papaevangelou, G., Sugg, U., Haas, H., Mushahwar, I. K., Ling, C.-M., Overby, L. R., Deinhardt, F. Detection of HBeAg and anti-HBe in acute hepatitis B by a sensitive radioimmunoassay. J. Med. Virol. 3:67-76, 1978.

22. Norkrans, G., Frosner, G., Iwarson, S. Determination of HBeAg by radioimmunoassay: prognostic implications in hepatitis B. Scand. J. Gastroenterol. 14:289-293, 1979.

23. Krugman, S., Overby, L. R., Mushahwar, I. K., Ling, C.-M., Frosner, G. G., Deinhardt, F. Viral hepatitis type B. Studies on natural history and prevention re- examined. N. Engl. J. Med. 300:101-106, 1979.



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