Vol. 17, No. 3 Printed in U.S.A.

INFECTION AND IMMUNITY, Sept. 1977, p. 550-554 Copyright © 1977 American Society for Microbiology

Expression of Hepatitis B Virus-Specific Markers in Asymptomatic Hepatitis B Surface Antigen Carriers G. HESS,' W. ARNOLD,' J. W.-K. SHIH,2 P. M. KAPLAN,2 R. H. PURCELL,3 J. L. GERIN 2* AND K. H. MEYER ZUM BUESCHENFELDE'

Second Department of Internal Medicine, University of Mainz, 6500 Mainz, Federal Republic of Germany'; Molecular Anatomy Program, Oak Ridge National Laboratory, Rockville, Maryland 208522; and Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 200143

Received for publication 11 April 1977

A study was undertaken to assess the state of hepatitis B virus infection in a of asymptomatic hepatitis B surface antigen (HB,Ag) carriers. This study confirmed that the presence of hepatitis B e antigen (HBeAg) in serum was closely associated with serum HB8Ag-specific deoxyribonucleic acid polymerase activity, hepatitis B core antigen (HBcAg) in serum and liver cell nuclei, and a histological picture of chronic hepatitis. No HB8Ag-specific deoxyribonucleic acid polymerase activity or HB,Ag was detected in highly concentrated anti-HBepositive sera. In addition, liver biopsy specimens from carriers with anti-HBe were negative for Hb,Ag by immunofluorescence, and the liver histology was either normal or revealed only fatty changes. These data indicate that the antiHBe-positive sera contained either no Dane particles or, if present, at least a 500fold-lower concentration of Dane particles than that found in HBeAg-positive group

sera.

The hepatitis B e antigen (HBeAg) and anti- and tested for HBV-specific DNA polymerase body (anti-HBe) systems were first described by activity and HB,Ag to determine whether Dane Magnius and Espmark (11, 12). It was demon- particles were present. strated that HBeAg is antigenically distinct PATIENTS AND METHODS from hepatitis B surface antigen (HB8Ag) (11, Patients and source of sera. Individuals found to 12) and hepatitis B core antigen (HBcAg) (20), be HB,Ag positive at the time of blood donation were but closely related to hepatitis B virus (HBV) retested 3 months later. Those who remained HB.Ag infection. Although the nature of the HBeAg is positive were considered HB8Ag carriers and followed still unknown, it is well established that the de- in a prospective study of the Deutsche Forschungsgetection of HBeAg in the serum is closely linked meinschaft. At the time of entrance into this study, to infectivity (1, 17), HBV-specific deoxyribonu- each patient received an intensive physical examinacleic acid (DNA) polymerase activity (1, 6, 13, tion, which included laboratory tests and a liver bi15, 16), the presence of Dane particles in the opsy. In all cases, the liver biopsy material was suffiserum (4, 15, 20), and HBcAg in the liver cell cient to allow both conventional light microscopy to nuclei (5, 14). Most investigators (1, 4-6, 14, 15, determine the histological diagnosis and immunofluorescence microscopy to study the intracellular locali20) report a negative correlation of the above zation of HB8Ag and HBcAg. The immunofluoresparameters with HB.Ag-positive sera that con- cence technique used has been described in detail (2). tain anti-HBe. There is, however, one report by For serological studies, larger serum quantities El Sheikh et al. (4) that describes five cases in were available from 22 of these asymptomatic HB,Ag which both anti-HBe and Dane particles could carriers. Seven were HBeAg positive, and 15 were be simultaneously detected in the serum. Mur- anti-HBe positive. All carriers were positive for phy et al. (14) found HBcAg in the liver cell HBeAg or anti-HBe for at least 6 months prior to nuclei in one anti-HBe-positive case. The latter bleeding. Assays. HBsAg was tested by radioimmunoassay observations led us to question whether the II, Abbott Laboratories). Unconcentrated techniques used were sensitive enough to dem- (Ausria were sera analyzed for HBeAg and anti-HBe by agar low levels of Dane onstrate particles in anti-HBe gel immunodiffusion as described by Magnius and sera. (11, 12). The agar used was slightly modifed Therefore, sera from HBeAg- and anti-HBe- Espmark as reported earlier (5). Reference sera, containing positive asymptomatic HB8Ag carriers, who both HBeAg components, were kindly provided by J. were biopsied and followed for at least 6 months 0. Nielsen, Copenhagen, Denmark. prior to this study, were highly concentrated Serum HB,Ag was tested by solid-phase radioim550

551

EXPRESSION OF HBV-SPECIFIC MARKERS

VOL. 17, 1977

phosphate-buffered saline (PBS), pH 7.4, containing 0.5% bovine serum albumin (BSA). As measured by DNA polymerase activity, an average of 60.7% of the total starting activity could be recovered in the 100fold concentrate by this procedure. Sucrose gradient analysis of serum concentrates. A 10-ml amount of one HBeAg positive serum (sample no. 1, Table 1) and one anti-HBe-positive serum (sample no. 11, Table 1) were layered on 20% (wt/wt) sucrose and centrifuged in an SW27 rotor at 20,000 rpm for 5 h at 40C. The pellets were suspended in 500 p1 of PBS-BSA and layered on top of a continuous 10 to 30% (wt/wt) sucrose gradient with a 1-ml cushion of 65% (wt/wt) sucrose. After centrifugation at 40,000 rpm for 2.5 h at 40C in an SW41 rotor, fractions were collected and assayed for DNA polymerase activity (75 pl), HBcAg (201ul), and HB.Ag (5 pl). Immunoelectron microscopic studies. Five-milliliter amounts of five sera (one HB.Ag positive, sample no. 1, Table 1, and four anti-HB. positive, sample no. 9, 12, 14, and 16, Table 1) were concentrated by pelleting through 20% (wt/wt) sucrose in an SW41 rotor at 40,000 rpm for 4 h at 40C. After suspending in 200 Ad of PBS, 20 pl of anti-HB. was added and reacted overnight at 40C. Samples were centrifuged in an SW50.1 rotor at 15,000 rpm for 30 min at 40C. After repeated washings with PBS, the pellet was

munoassay as described by Purcell et al. (18). Results were expressed as positive/negative (P/N) ratios or as counts per minute.

HBV-specific DNA polymerase activity was measured in concentrated and unconcentrated sera by the method of Kaplan et al. (9). Results were expressed in counts per minute. The data presented in this paper were calculated by subtracting the counts per minute of an HB.Ag-negative normal human serum from the counts per minute obtained from the test sample. Values higher than 30 cpm (5 standard deviations above the mean value of normal human serum) were regarded as positive. Unconcentrated sera that were positive for polymerase activity were tested for specificity as described by Kaplan et al. (8). Concentration of Dane particles. Dane particles present in 10 mil of serum from each individual were concentrated in the following manner. After a lowspeed centrifugation (2,000 x g, 15 min), the serum was layered on a discontinuous gradient composed of 2.5 mi of 65% (wt/wt) sucrose and 25.5 ml of 20% (wt/wt) sucrose. After centrifugation in an SW27 rotor at 40C for 5 h at 27,000 rpm, the interface was harvested, diluted, and recentrifuged as above. The interface was again collected, diluted to 10 ml, layered on a 2-ml cushion of 20% (wt/wt) sucrose, and pelleted in an SW41 rotor by centrifuging at 40,000 rpm for 4 h at 40C. The pellet was finally suspended in 100 ,l of

TABLE 1. Serological, histological, and inmunofluorescence analyses of material obtained from 22 asymptomatic HB.Ag carriers

No. Sex

Age

M F M M M

HB A HB.Ag9

54 62 53 36 15 40 28 36 20 29 34 33 25 21 27 39 53

+ +

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

M F M

M M M M F M M M M M F F F F

25

21 37 29 37

-

-

+ + + +

+ + + + + + + + + +

-

-

Immunofluores-

(lOOx)

cence

Anti- DNA-P- DNA-Pa HBA (P/N) (cpm) HBe (cpm)

+

-

Concentrated sera

+ + + +

+

350 52 199 201 295 347 70 25 27 -16

18,t34

1

27 9 -15 11 -20 21

-27 -34 -40 -41 -13 0 -10

260 100 72 30 38 95 26 1.1 1.2 0.8 1.0 1.0 0.9 0.8 1.0 0.9 1.2

25 15 -15 -8 -17

3 7 -37 -34 -24

0.9 1.1 0.9 0.7 2.8

3,939 14,405 15,986 14,761 18,741 2,827 -9 -34 -29

Liver histol- SGOT

HBAg HB.Ag -

+ +

+

+

+

+ + + +

+

-

-

+ + + +

-

+

-

-

-

+ + +

+ + + +

+ +

OgYC

CPH CAH CPH CAH CPH CAH CPH NL Fibrosis

TCN FC FC NL CPH FC FC FC FC NL FC NL NL

SGPT

(MU/M)(MU/Ml)d

114 35

27 60 34 32 26 14 9 12 12 14 8 23 24 10 14 14 19 10 9 10

154 39 38 138 42 69 39 14 8 17 7 15 7 39 29 10 16 15 28 20 10 8

DNA-P, DNA polymerase activity; normal value,

Expression of hepatitis B virus-specific markers in asymptomatic hepatitis B surface antigen carriers.

Vol. 17, No. 3 Printed in U.S.A. INFECTION AND IMMUNITY, Sept. 1977, p. 550-554 Copyright © 1977 American Society for Microbiology Expression of Hep...
763KB Sizes 0 Downloads 0 Views