THE JOURNAL OF INFECTIOUS DISEASES. VOL. 136, NO.4. OCTOBER 1977 © 1977 by the University of Chicago. All rights reserved.

Hepatitis B e Antigen and Infectivity of Hepatitis B Virus T. Shikata, T. Karasawa, K. Abe, T. Uzawa, H. Suzuki, T. Oda, M. Imai, M. Mayumi, and Y. Moritsugu

From the First Department of Pathology, Nihon University School of Medicine; the First Department of Internal Medicine, University of Tokyo School of Medicine; the Immunology Division, [ichi Medical School; the Hepatitis Division, the Tokyo Metropolitan Institute of Medical Science; and the Enterovirus Division, National Institute of Health, Tokyo, Japan

Hepatitis B virus (HBV) mainly related to the blood is transmitted from person to person by transfusion of blood and its products, although the modes of transmission appear multiple and diverse, including vertical transmission from a mother to her child. Recent advances in hepatitis research have disclosed much about HBV and HBV infection with increasing complexity. Three antigen-antibody systems associated with HBV infection have been introduced. Hepatitis B surface antigen (HB s Ag) and hepatitis B core antigen (HB c Ag) and antibody to each (anti-Hb, and anti-Hfs., respectively) are relatively well understood. The third antigen-antibody system is composed of the e antigen of HBV (HB e Ag) and its antibody (anti-HB e) [IJ; much investigation is needed to clarify its nature and role in HBV infection. Several important observations associated with the e system included the relation

of the e system to the infectivity of HB s Ag-positive serum: exclusive occurrence of vertical HB s Ag transmission from an HB e Ag-positive mother to her children but not from an anti-Hb.positive mother to her children [2J and horizontal transmission of hepatitis B by needle-stick giving the similar correlation [3J. In line with these observations, HBV-associated DNA polymerase, abundant Dane particles, and higher titers of HB s Ag are the usual findings in HB s Ag-positive sera also positive for HB e Ag but not for HB s Ag-positive sera also positive for anti-H'B, [3-5]. Thus HB e Ag-positive serum seems to be more infectious than anri-Hb.-positive serum. To prove this relation between infectivity of HBV and the e system, we infected chimpanzees with HBV. Infectivity titrations of HB s Agjadr-positive sera differing in the e system were also attempted. The direct relationship between the dose of the inoculated virus and the incubation period before appearance of HB s Ag in the blood and elevation in level of serum glutamic-pyruvic transaminase (SGPT; also known as alanine aminotransferase) is also described.

Received for publication March 7, 1977, and in revised form May 16, 1977. This study was supported by a grant from the Japanese Ministry of Health and Welfare. We thank Drs. K. Koshimizu and N. Yoshihara for their cooperation. Please address requests for reprints to Dr. Toshio Shikata, the First Department of Pathology, Nihon University School of Medicine, 30 Kamimachi Oyaguchi Itabashi-ku, Tokyo, 173 Japan.

Materials and Methods

Chimpanzees. Seven chimpanzees (five males and two females) born in West Africa and ob-


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For confirmation of the difference in the infectivity of hepatitis B surface antigen (HB s Ag)-positive serum according to differences in the e antigen system, four chimpanzees were inoculated with serum positive for hepatitis B e antigen (HB e Ag), and three chimpanzees were inoculated with serum positive for antibody to HB e Ag (anti-Hb). Since the infectivity titrations are not yet completed, the end infectivity titer of each serum is not known. All four chimpanzees given injections of 10-1, 10-4 , or 10-8 dilutions of HB e Ag-positive serum developed hepatitis B virus infection, whereas the one chimpanzee injected with undiluted anti-HBe-positive serum became infected, and other chimpanzees injected with diluted anti-Hlsj-positive sera did not. As judged from the length of the incubation period before appearance of HB s Ag in blood, there seemed to be a remarkable difference in infectivity between the HB e Ag-positive serum and the anti-Hls.-positive serum; the former serum was 108 times more infectious than the latter.


Shikata et al.

Ag were collected in an attempt to equalize HB s Ag titers among the four sera, because the titer of HB s Ag in serum with anti-H'B, is generally quite low. Sera no. 2 and 4 were diluted with fetal calf serum. Volumes of I ml of serum were injected iv into the chimpanzees. No immunosuppressive agent was administered before or after inoculation prior to the appearance of HB s Ag. After the appearance of HB s Ag, however, cyclophosphamide was injected iv into chimpanzees no. 5 and 6 to establish the chronic HB s Ag carrier state. Analysis of blood and serologic determinations. After inoculation each chimpanzee was bled every week for determinations of HB s Ag (by reverse passive HA and radioimmunoassay), antiHB s (by passive HA), and anti-Hb, (by immune adherence HA), SGPT, SGOT (serum glutamic-oxalacetic transaminase or aspartate aminotransferase), alkaline phosphatase, bilirubin, total cholesterol, zinc sulfate turbidity, serum protein, immunoglobulins, and complement. With the appearance of HB s Ag, large volumes of serum were obtained by plasmapheresis for detection of HB e Ag, anti-HB e , HB Ag-specific DNA polymerase, and Dane particles. Histopathology. Liver aspiration biopsy was performed every other week. Cylinders of liver were processed for examination by light microscopy (including orcein staining), electron microscopy, and immunofluorescence for detection of HB s Ag and HB e Ag. Unfortunately, the experiment was interrupted by the accidental deaths of chimpanzees no. 5, 6, 9, and 14 due to a malfunction in the ventilator machine. Chimpanzees no. 5 and 6 died 33 weeks after inoculation, and no. 9 and 14 died 20 weeks after inoculation. Postmortem

Table 1. Characteristics of sera containing hepatitis B antigens used for inoculation of chimpanzees. Serum no. 1 2 3 4

Designation ]HB-002-K-2765 ]HB-OOI ]HB-OI2-3-6288 ]HB-OOI




16-year-old male Pooled 22-year-old male Pooled





HB s Agl adr* 2 14 2 15 2 13 2 14

e systernf HB e Ag HB e Ag Anti-HBe Anti-HB e

DNA polymerase I 171 299 29


NOTE. SGPT = serum glutamic-pyruvic transaminase; SGOT = serum glutamic-oxalacetic transaminase; HB s Ag = hepatitis B surface antigen; HB e Ag = hepatitis B e antigen; anti-Hls., = antibody to HB e Ag. *Titrated by the reverse passive HA method. tDetermined by immunodiffusion. +Expressed as the difference (cpm) between the uptake of [3H] thymidine triphosphate by a sample pretreated with normal rabbit serum and that by a sample pretreated with rabbit antibody to HB s Ag,

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tained through Primate Imports, Port Washington, N.Y., were used. They were three to four years old and weighed 15-30 kg. Before importation, two tests for HB s Ag, anti-Hb., and anti-Hfs., with an interval of three weeks, were negative for each animal. After arrival in Japan the chimpanzees were caged individually and held for about two months for quarantine and adaptation. Intestinal parasites and microfilaria were found in the blood of most of the animals. All were nonreactive for tuberculosis, and no evidence of hepatitis was present as detected by liver function tests and histopathologic study by aspiration needle biopsy. Before the start of the experiment, each animal in a squeezing cage was individually put in an isolator made of stainless steel. Inoculation, bleeding, and liver biopsy were performed on a table outside the isolator with particular care paid to the protection against contamination among the chimpanzees. During the period of observation, no evidence of HBV infection developed in one control animal, and no booster response of anti-Hb, or anti-Hls, was seen in chimpanzees that had developed antiHB e and anti-Hls, after successful experimental infection. Inocula. Four infectious sera containing high titers of HB s Agj adr were used (table 1). Sera no. I and 2 were positive for HB e Ag and HB Ag-associated DNA polymerase. Serum no. I was from a 16-year-old asymptomatic HB s Ag carrier. No.2 was pooled serum from more than 10 similar HB s Ag carriers. Serum no. 3 was from a 22-year-old asymptomatic HB s Ag carrier positive for anti-Hb., No.4 was pooled serum from a number of asymptomatic HB s Ag carriers positive for anti-Hb., For sera no. 3 and 4, anti-H'Bi-positive sera with the highest possible titers of HB s


HB e Ag and Infectivity of HBV

liver specimens were taken from chimpanzees no. g and 14 and were frozen at -70 C within 1 hr of the animals' deaths. The specimens were studied for HB s Ag and HB c Ag by immunofluorescence. Fluorescein isothiocyanate-labeled y-globulin was prepared from rabbit anu-Hb.. AntiHB c prepared from the serum of an HB s Ag carrier was conjugated with fluorescein isothiocyanate. Results

Table 2. Summary of data on chimpanzees inoculated with sera containing hepatitis B antigens. Week after inoculation indicated finding detected in serum Animal Inoculated no. serum no. * 1 10 5 6 2 9§ 14§

1 2 2 2 3 4 4



Undiluted 10- 1 10-4

1 4-7 8-25 13-29 19-20



HBe Ag

Anti-HBst Anti-HB c t 7 11

11-17 16-25 21

20 13 14 19 21

SGPT elevation +

7-9 (378) 12-24 (690) 18-26 (460)

Hepatic histopathology

+ + +

10.. .2 10-5

NOTE. HB s Ag = hepatitis B surface antigen; HB e Ag = hepatitis B e antigen; anti-Hb, = antibody to HB s Ag; anti-Hls.. = antibody to hepatitis B core antigen; (+) = present; (-) = absent; leaders indicate finding not detected. *All sera were HB s Agjadr-positive. Serum no. 1 was HBe Ag-positive and DNA polymerase-positive. No.2 was pooled HBe Agand DNA polymerase-positive serum. Serum no. 3 was anti-Hlig-positive and DNA polymerase-negative. No.4 was pooled antiHBe-positive, DNA polymerase-negative serum. tPositive for week indicated and thereafter. +Numbers in parentheses are maximal values for serum glutamic-pyruvic transaminase (SGPT) in Karmen units. § These chimpanzees had no evidence of hepatitis B virus infection for up to 20 weeks after inoculation.

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Chimpanzee no. 1 (table 2) was inoculated with I ml of undiluted serum no. 1 (HBe Ag-positive). Chimpanzees no. 10, 5, and 6 received 1-ml inocula that consisted of 10-1, 10-4 , and 10-8 dilutions of HB e Ag-positive pooled serum (no. 2), respectively. HB s Ag was detected in the blood of chimpanzee no. 1 only once, at seven days after inoculation; on this occasion the animal's serum was positive by radioimmunoassay and negative by reverse passive HA. There might be a question as to whether this HB s Ag in the blood was the original HB s Ag inoculated and not cleared from the blood or HB s Ag that had multiplied within liver cells and was released into the blood. Chimpanzee no. 2, however, which received the same dose of HB s Ag, did not have HB s Ag in the blood on the same day after inoculation. In chimpanzee no. 1 anti-Hls, and anti-HB c developed seven and 20 weeks after inoculation, respectively, signifying HBV infection in the ani-

mal. No laboratory or morphological evidence of hepatitis was found. Chimpanzees no. 10 (figure 1, top), 5 (figure 1, bottom), and 6, which were inoculated with 10-1 , 10-4 , and 10-8 dilutions, respectively, of HB e Ag-positive pooled serum, developed HB s antigenemia four, eight, and 13 weeks after inoculation, respectively. Thus a reverse linear relationship was seen between the dilution of the sera (dose of the virus) and the incubation period for appearance of HB s Ag (figure 2): the higher the dose of virus, the shorter the incubation period. A similar relationship was noted between the dose of virus inoculated and the incubation period before elevation of levels ofSGPT. Chimpanzee no. 10 (figure 1, top) inoculated with the 10-1 dilution of serum no. 2 developed typical type B hepatitis. The animal became HB s Ag-positive four weeks after inoculation, and abnormalities in liver function were detected seven weeks after inoculation. The highest level of SGPT seen at week 8 was 378 units. The titer of HB s Ag peaked when the level of SGPT started to rise, and HB s Ag was no longer present at week 10. Anti-Hls, and anti-Hls, developed during weeks 11 and 13, respectively. After the appearance of HB s Ag, chimpanzees no. 5 and 6 were started on a regimen of cyclophosphamide (100 mg iv twice a week). HB s antigenemia had persisted for 16 and 17 weeks, respectively, in the two chimpanzees but disappeared subsequently in both. HB e Ag was detected in these animals (no. 5, from week 11 to week 17; no. 6, from


Shikata et al.

600 15

400 10

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Anti HBs





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Hepatitis B e antigen and infectivity of hepatitis B virus.

THE JOURNAL OF INFECTIOUS DISEASES. VOL. 136, NO.4. OCTOBER 1977 © 1977 by the University of Chicago. All rights reserved. Hepatitis B e Antigen and...
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