INFECTION AND IMMUNITY, Sept. 1976,

Vol. 14, No. 3

p. 631-635

Printed in U.S.A.

Copyright C 1976 American Society for Microbiology

Hepatitis B Antigen-Associated Deoxyribonucleic Acid Polymerase Activity and e Antigen/Anti-e System MITSUNOBU IMAI, FUMIKO C. TACHIBANA, YASUO MORITSUGU, YUZO MIYAKAWA, AND MAKOTO MAYUMI* Immunology Division, Jichi Medical School, Tochigi-ken 329-04; Department of Enteroviruses, National Institute of Health, Musashi Murayama, Tokyo 190-11; the Third Department ofInternal Medicine, University of Tokyo, Tokyo 113; and Hepatitis Division, the Tokyo Metropolitan Institute of Medical Science, Bunkyo-ku, Tokyo 113, Japan*

Received for publication 30 April 1976

Serum samples of 403 asymptomatic blood donors carrying hepatitis B surface antigen (HB,EAg) were concentrated threefold and tested for e antigen and antibody to e antigen (anti-e) by immunodiffusion. Hepatitis B antigen (HBAg)associated deoxyribonucleic acid (DNA) polymerase activity was specifically determined by the difference in incorporation of [methyl-3H]thymidine 5'-triphosphate into DNA by an aliquot of centrifuged serum samples after it had been treated either with normal rabbit serum or with rabbit antibody to

HB.Ag. All of 58 serum samples containing e antigen revealed HBAg-associated

DNA polymerase activity, whereas none of 96 samples containing anti-e did. In the remaining 249 samples in which neither e antigen nor anti-e was found, 62 showed specific DNA polymerase activity, although at lower levels than the samples containing e antigen.

Among three morphologically different enti- designed to find the relationship between ties of hepatitis B antigen (HBAg), i.e., 42-nm HBAg-associated DNA polymerase activity and double-shelled Dane particles (3) and 20-nm the e antigen/anti-e system. spherical and tubular antigens, only Dane parMATERIALS AND METHODS ticles contain deoxyribonucleic acid (DNA) (16, Serum samples. At regional centers of the Japan 17). In addition, DNA polymerase activity has Cross Association, healthy blood donors were been detected in preparations containing hepa- Red screened for HB%Ag by the immune adherence hetitis B surface antigen (HB,Ag) (4) and has magglutination (IAHA) method (11), and a total of been found specifically associated with Dane 403 HB,Ag-positive serum samples were obtained. particles (3a). Based upon these observations, They were tested for all the immunological markers Dane particles have been considered to repre- of HBV infection presently available, i.e., titer and sent the putative hepatitis B virus (HBV). subtype of HB,Ag, titers of HB,Ag and antibody to Dane particles are also found to be associated HB,Ag (anti-HB,), and the presence of e antigen with antigenic determinants by which they are and anti-e, as well as for HBAg-specific DNA polymdiscriminated from HBAg of other entities; the erase activity. Serological tests. HB%Ag was determined and ticore of Dane particles shows a distinct anti- trated by reverse passive hemagglutination. The genic activity designated as hepatitis B core test serum serially diluted twofold in phosphate antigen (HBCAg) (1, 5), and recently the e anti- buffer (0.01was M, pH 7.6) containing 0.1 M NaCl and gen of Magnius and Epsmark (10) has also been 1% normal horse serum in a V-bottom microtiter identified on the surface of Dane particles (13). plate (Cooke Engineering Co., Alexandria, Va.). These immunological markers may be of use in One drop (25 gd) of glutaraldehyde-fixed sheep identifying Dane particles and in labeling erythrocytes coated with horse antibody to HB%Ag HB,Ag-positive serum samples with high infec- (anti-HB,; dominant specificity anti-a [9]) was added tivity if their mutual relationship can be estab- to each well; horse anti-HE, was specifically purified an affinity column of HB%Ag. After incubation lished. We have found that HB,Ag-positive se- using at room temperature for 60 min, the pattern of sedirum samples containing e antigen are invaria- mentation observed. The titer of HB%Ag was bly associated with HBCAg activity, whereas expressed aswas the highest dilution at which hemagthose containing antibody to e antigen (anti-e) glutination was observed. are not (K. Takahashi, M. Imai, F. Tsuda, T. Subtyping of HB%Ag was performed by the heTakahashi, Y. Miyakawa, and M. Mayumi, J. magglutination inhibition method (6). Briefly, the Immunol., in press). The present study was test serum was serially diluted twofold in a microti631

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IMAI ET AL.

ter plate, and 1 drop of buffer containing 2 hemagglutinating units of monospecific antibody directed to either a, d, y, w, or r determinant (2, 8) was delivered to each well. After incubation, 1 drop of a suspension of sheep erythrocytes coated with HB.Ag of the corresponding specificity was added. Presence of the antigenic determinant was detected by the failure of indicator cells to agglutinate. Anti-HB, was determined by the IAHA method, using HB,Ag that had been isolated from plasma of asymptomatic carriers as has been described previously (18). HBCAg activity in the serum sample was also determined by IAHA. Dane particles in 1 ml of the test serum were precipitated by adding 0.5 ml of rabbit anti-HB% (dominant specificity anti-a; passive hemagglutination titer 1:1,280) to separate them from any anti-HB, present. The precipitate was suspended in 50 ul of buffer containing Nonidet P-40, 2mercaptoethanol, and Pronase E (Kaken Kagaku, Tokyo, Japan) to expose antigenic sites of HBCAg, and was then titrated by a microtiter technique. e antigen and anti-e were determined in the test serum, which had been concentrated threefold by adding 50 mg of Lyphogel (Gelman Instruments Co., Ann Arbor, Mich.) per ml. Ouchterlony double immunodiffusion was performed in wells (3 mm in diameter, cut 3 mm apart [edge to edge]) in 0.9% agarose dissolved in 0.01 M tris(hydroxymethyl)aminomethane (Tris) buffer (pH 7.6) containing 0.1 M NaCl, 2% dextran T-500, and 20 mM ethylenediaminetetraacetic acid. Serum samples containing e antigen and anti-e were kindly supplied by Lars 0. Magnius of Statens Bacteriologiska Laboratorium, Stockholm, and by Jay H. Hoofnagle of the Bureau of Biologics, Bethesda, Md., and were used as reference reagents. Reagents for DNA polymerase assay. Deoxyadenosine 5'-triphosphate (dATP), deoxycytidine 5'-triphosphate (dCTP), and deoxyguanosine 5'-triphosphate (dGTP) were purchased from Calbiochem (San Diego, Calif.). [methyl-3H]thymidine 5'-triphosphate ([3H]TTP), with a specific activity of 50 Ci/mmol, was obtained from New England Nuclear Corp. (Boston, Mass.). Determination of DNA polymerase activity. DNA polymerase activity in the test serum was determined by the modified method (12) originally described by Kaplan et al. (7). Before the test, Dane particles in the serum were concentrated in order to increase the sensitivity. One milliliter of serum was centrifuged at 6,600 x g for 20 min. The supernatant was further centrifuged at 59,000 x g for 3 h. The precipitate was suspended in 0.1 ml of Tris-hydrochloride buffer (pH 8.0) containing 0.1 M NaCl, 1% bovine serum albumin and 0.1% NaN3. Fifty microliters of the sample, which was concentrated 1:10, was mixed with either 25 j.d of a 1:25 dilution of rabbit anti-HB, (dominant specificity anti-a; original passive hemagglutination titer 1:40,000) or the same amount of a 1:25 dilution of normal rabbit serum. After incubation for 1 h at 37°C, 25 ,ul of a 1:20 dilution of goat anti-rabbit gamma globulin antiserum was added to each sample, and the samples were further incubated at 37°C for 1 h. They were then spun at 600 x g for 30 min, and the supernatant was removed. A 50-,u amount of the

INFECT. IMMUN. supernatant was mixed with 25 ,ul of the reagent solution. The final concentration of the reagents in the reaction mixture was 0.2 mM each of dATP, dCTP, and dGTP; 0.15 ,uM [3H]TTP (0.375 ,Ci/50 ,ul); 30 mM MgCl2; 200 mM KCI; 25 mM 2-mercaptoethanol; 0.2% Nonidet P-40; and 50 mM Tris (pH 8.0). After incubation at 37°C for 3 h, the reaction mixture was spotted on a diethylaminoethyl-cellulose disc (Whatman DE81), washed successively with 5% Na2HPO, solution, distilled water, and ethanol, and dried. Radioactivity retained was measured in a scintillation counter with scintillation fluid consisting of 0.1 g of 1,4-bis[2-(5-phenyloxazolyl)]benzene and 5.0 g of 2,5-diphenyloxazole per liter of toluene. The count of the sample pretreated with normal rabbit serum minus that of the sample pretreated with rabbit anti-HB was calculated and used as a measure of HBAg-associated DNA polymerase activity.

RESULTS The results of DNA polymerase assay and the prevalence of e antigen and anti-e in serum samples of 403 asymptomatic blood donors carrying HB,Ag are summarized in Table 1. The count of 20 normal serum samples without HBsAg or anti-HB,, treated in an identical manner as in the assay of HBAg-specific DNA polymerase activity, fell within the range of -32 to 10 cpm (mean ± standard deviation, 0.1 ± 7.2). e antigen was detected in 58 samples (14.4%), and anti-e was detected in 96 (23.8%). All the samples with e antigen showed specific DNA polymerase activity ranging from 30 to 626 cpm. In sharp contrast, none of 96 samples with anti-e revealed any detectable DNA polymerase activity (-0.7 ± 8.0 cpm). There were 249 samples in which neither e antigen nor anti-e was identified by immunodiffusion. Specific DNA polymerase activity was detected in 62 of them, although the count was relatively low (range, 30 to 69 cpm). Consequently, DNA polymerase activity was demonstrated in a total of 120 (29.8%) of 403 HBsAg-positive serum samples tested. Fifty serum samples containing e antigen and 50 containing anti-e were randomly selected and further tested for titer and subtype of HB,Ag, as well as for titers of HBCAg and antiHB,. The uptake in cpm of [3H]TTP by the sample pretreated with normal rabbit serum and that by the sample pretreated with rabbit anti-HIB,, and their difference, which represented HBAg-specific DNA polymerase activity, are given in Tables 2 and 3. Serum samples with e antigen not only showed DNA polymerase activity, but they also invariably revealed HBCAg activity. In comparison, samples with anti-e did not contain any detectable DNA polymerase activity, nor did

HBAg-ASSOCIATED DNA POLYMERASE AND e ANTIGEN

VOL. 14, 1976

TABLE 1. Relationship ofHBAg-associated DNA polymerase activity and the e antigenlanti-e system in serum samples of asymptomatic carriers of hepatitis B surface antigen e

antigen or anti-e

Positive'

DNA polymerase Negative Total (%)

58 e antigen 0 58 (14.4) Anti-e 0 96 96 (23.8) _b 62 187 249 (61.8) Total (%) 120 (29.8) 283 (70.2) 403 (100.0) I Specific uptake of [3H]TTP precipitable by antiHB, of 30 cpm or more. ' Neither e antigen nor anti-e was detected by immunodiffusion.

they reveal any detectable HBeAg activity. In general, samples with DNA polymerase activity and e antigen showed higher titers of HB,Ag. The log2 geometric mean titer for 50 samples with e antigen was 11.90 + 1.42, significantly higher than the value of 50 samples with anti-e at 9.96 + 1.97 (t test, P < 0.001). However, there was a considerable overlapping of the titers between these two groups. Subtypes of HBAg had no relation to DNA polymerase activity; both HB,Ag/adw and HB,Ag/adr were found in each group. It is also apparent from these tables that the HBAg-positive serum samples containing e antigen and HBAg-specific DNA polymerase activity did not necessarily reveal higher titers of anti-HB, than those. containing anti-e, but were devoid of specific DNA polymerase activity (10.96 + 2.08 versus 11.56 + 2.20, t test, P < 0.1). DISCUSSION The association of HBAg-specific DNA polymerase activity with e antigen in HB,Ag-positive serum samples has been established. The results reported by Nodenfeld and Kjellen for a limited number of samples seem to reveal the same tendency (14). Moreover, the antigenic activity of the core of Dane particles (HBCAg) was also found to be associated with DNA polymerase activity, further making clear the relationship of e antigen and HBV. The HB,Agpositive serum samples containing anti-e, however, showed neither DNA polymerase activity nor any detectable HBCAg activity. Based on these observations, there appear to be two kinds of HBr, antigenemia, one containing e antigen as well as HBV-specific DNA polymerase activity and HB,Ag, and the other containing anti-e but devoid ofenzymatic and immunological tags of Dane particles. However, the other immunological markers of HBV infection, namely HB,Ag and anti-HB,, did not ap-

633

TABLE 2. HBAg-specific DNA polymerase activity in 50 serum samples of asymptomatic carriers containing e antigen [3H]TTP uptake of HBAgA,ntisample specific HB,Agg HB~Ag HB, treated with: DNA titer titer polymer(log2) ase (Ilog,)P Nor- AntiTiter Submal HB, activity (log2) type serum

adw 626 14 4 635 9 14 adr 3 536 589 53 8 adr 510 12 5 498 14 500 11 adr 3 489 12 484 adr 494 10 5 432 10 462 30 adr 5 adr 11 5 421 81 340 adw 5 327 12 392 65 adr 323 8 358 35 5 adr 5 320 12 360 40 adr 318 12 6 385 67 adr 5 12 360 48 312 adr 12 5 311 14 297 adr 12 5 322 43 279 adw 3 213 13 247 34 adr 11 5 207 288 81 adr 4 13 197 6 191 adr 7 4 199 11 188 adw 5 164 10 174 10 adw 3 11 203 41 162 adr 11 6 176 15 161 adr 13 6 158 239 81 adr 5 12 164 156 8 adr 12 167 15 5 152 13 adr 7 9 152 8 144 12 adr 13 161 5 19 142 11 adr 4 11 219 78 141 13 adr 6 6 149 14 135 11 adw 4 8 163 33 130 11 adr 4 10 150 25 125 11 adr 12 5 158 41 117 13 adr 6 9 116 120 4 12 adw 6 12 175 61 114 12 adr 5 11 134 21 113 10 adr 5 112 11 123 11 16 adr 5 8 127 16 111 12 adr 6 6 167 66 101 11 adr 6 14 100 92 8 104 17 7 12 adr 4 87 11 adr 6 11 83 109 26 12 9 adr 7 8 89 81 13 adr 10 5 104 23 81 4 10 adr 12 92 20 72 8 adr 13 77 8 5 69 10 adr 147 87 5 13 60 13 adr 13 65 21 5 44 12 adr 6 6 64 23 41 11 adr 4 13 130 95 35 11 adr 10 2 5 32 34 11 adr 5 11 37 7 30 a Log2 geometric mean titer + standard deviation, 11.90 + 1.42. b L0g2 geometric mean titer + standard deviation, 10.94 + 2.20. 11 13 14 13 13 14 13 13 13 13 13 13 10 13 11 12 12 13 11 12 13 11 12 10

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IMAI ET AL.

TABLE 3. HBAg-specific DNA polymerase activity in

INFECT. IMMUN.

pear to indicate much about the presence of Dane particles. The log2 geometric mean titer of anti-HB, of the samples with e antigen, HB,Ag, and DNA polymerase activity was not uptake of HBAgdifferent from that of those containing anti-e Antisample specific HB,Ag but no HB,Ag or DNA polymerase activity. HIB,Ag HB, treated with: DNA titer titer Norpolymer- Although the former had a significantly higher Titer Subase (10g2)b mal Antimean titer of HB3Ag than the latter on the (log2)a type serum HB, activity average, a large overlap between these two -e 8 adr 12 24 8 situations made it difficult to assess the pres16 10 adr 13 57 42 12 ence of Dane particles based on the titer of 13 adr 12 20 11 9 HB5Ag alone. 12 adw 14 15 7 8 Since, at the present moment, it is clear that 11 adr 13 14 8 6 there are at least two kinds of HB5 antigene13 adr 10 10 5 5 mia, one with all the immunochemical activi9 adr 10 29 25 4 ties characteristic of HBV and the other with13 adw 14 4 10 6 out, it would be natural to determine the differ12 adr 14 6 4 10 ence in infectivity between these two situa11 adr 14 8 4 4 11 adr 8 4 tions. We have noted that asymptomatic car11 4 10 adr 14 12 9 3 riers of HB.Ag with e antigen in their serum 11 adr 6 11 8 3 invariably transmitted HBV to their infants, 11 adr 12 10 7 3 whereas those with serum anti-e did not (15). 7 adr 9 5 2 3 Based on these observations, it has been postu11 adr 14 10 8 2 lated that e antigen may be used as a predictor 12 adr 12 8 6 2 of vertical transmission of HBV. The present 9 adr 12 8 9 1 finding that HBAg-specific DNA polymerase 9 adr 10 8 1 7 activity was associated with e antigen, coupled 9 adr 6 10 5 1 8 adr with the observation that the antigenic activity 12 4 1 5 5 adr 1 10 5 4 of the core of Dane particles was also associated 8 adr 34 11 34 0 with it, strengthens this view. 8 adr 11 34 34 0 A significant activity of HBAg-associated 9 adr 11 17 17 0 DNA polymerase was found even in some of the 13 adr 11 0 10 10 HB2Ag-positive samples in which neither e an7 adw 14 8 8 0 tigen nor anti-e was identified by immunodiffu11 adr 8 13 8 0 sion. Accordingly, a total of 120 (29.8%) of 403 4 10 adr 10 4 0 9 adr 14 -1 9 HB,Ag-positive samples showed DNA polymer10 5 adr -1 ase activity, at a rate considerably higher than 13 7 8 12 adr 12 8 -1 7 that of e antigen (58/403 or 14.4%). It would be 10 adr 8 4 -1 5 possible to detect more HBAg-specific DNA po9 adr -2 7 51 53 lymerase activity by applying a larger amount 9 adr 14 -2 12 14 of serum sample than was used in the present 12 adr 11 7 10 -3 since Dane particles can easily be experiments, 8 adw 9 3 6 -3 concentrated. Since presently no sensitive 7 adr 14 8 12 -4 methods are available for the detection of e 12 adr -4 6 10 10 antigen, such as radioimmunoassay and im9 adr 4 -4 10 8 10 adr -6 11 14 mune adherence hemagglutination, applicabil20 11 adr -6 12 9 15 ity of e antigen as an indicator of Dane particles 10 adr 13 3 -6 9 is hampered by the low sensitivity inherent to 9 adr 12 -7 22 15 immunodiffusion. HBAg-specific DNA polym12 adr 14 -7 13 20 erase activity in the serum, therefore, is un11 adr -7 9 8 15 useful as a more sensitive marker of doubtedly 12 adr 12 3 10 Dane particles than is e antigen. Anti-e, on the -7 8 adr 10 3 10 other hand, would be very valuable as the only 11 adr 12 -10 8 18 available indicator to denote the absence of 11 adr -32 12 34 66 a Log2 geometric mean titer + standard deviation, Dane particles. There were asymptomatic carrier mothers in 9.96 + 1.97. ° Log2 geometric mean titer + standard deviation, our series whose serum did not show either e 11.56 1.95. antigen or anti-e by immunodiffusion. Some of c No HBcAg was detected by immune adherence them vertically transmitted HBV to their chilhemagglutination. dren, whereas others did not. We are now ex50 serum samples of asymptomatic carriers containing anti-e [3H]TTP

VOL. 14, 1976

HBAg-ASSOCIATED DNA POLYMERASE AND

tending the results described in the present paper and are looking for HBAg-specific DNA polymerase activity in the concentrated serum samples of those mothers to find its correlation, if any, with vertical transmission of HBV to their children. ACKNOWLEDGMENTS We thank the Japan Red Cross Association for supplying serum samples of asymptomatic HBAg carriers. This investigation was supported in part by grants from the Tokyo Metropolitan Government and the Japanese Ministry of Health and Welfare. LITERATURE CITED 1. Almeida, J. D., E. J. Rubenstein, and E. J. Stott. 1971. New antigen-antibody system in Australia-antigenpositive hepatitis. Lancet 2:1225-1227. 2. Bancroft, W. H., F. K. Mundon, and P. K. Russell. 1972. Determination of additional antigenic determinants of hepatitis B antigen. J. Immunol. 109:842848. 3. Dane, D. S., C. H. Cameron, and M. Briggs. 1970. Virus-like particles in serum of patients with Australia-antigen-associated hepatitis. Lancet 1:695-698. 3a. Greenman, R. L., and W. S. Robinson. 1974. DNA polymerase in the core of the human hepatitis B virus candidate. J. Virol. 13:1231-1236. 4. Hirschman, S. Z., S. J. Vernace, and F. Schaffner. 1971. DNA polymerase in preparations containing Australia antigen. Lancet 1:1099-1103. 5. Hoofnagle, J. H., R. J. Gerety, and L. F. Barker. 1973. Antibody to hepatitis-B-virus core in man. Lancet 2:231-243. 6. Imai, M., Y. Yamashita, Y. Miyakawa, and M. Mayumi. 1974. Hemagglutination inhibition assay of the common determinants and subspecificities of Australia antigen. Immunology 27:871-878. 7. 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.

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12:995-1005. 8. Le Bouvier, G. L. 1971. The heterogeneity of Australia antigen. J. Infect. Dis. 123:671-675. 9. Levene, C., and B. S. Blumberg. 1969. Additional specificities of Australia antigen and the possible identification of hepatitis carriers. Nature (London) 221:195196. 10. Magnius, L. O., and J. A. Epsmark. 1972. New specificities in Australia antigen positive sera distinct from Le Bouvier determinants. J. Immunol. 109:10171021. 11. Mayumi, M., K. Okochi, and K. Nishioka. 1971. Detection of Australia antigen by means of immune adherence hemagglutination test. Vox Sang. 20:178-181. 12. Moritsugu, Y., J. W. M. Gold, J. Wagner, R. Y. Dodd, and R. H. Purcell. 1975. Hepatitis B core antigen: detection of antibody by radioimmunoprecipitation. J. Immunol. 114:1792-1798. 13. Neurath, A. R., C. Trepo, M. Chen, and A. M. Prince. 1976. Identification of additional antigenic sites on Dane particles and the tubular forms of hepatitis B surface antigen. J. Gen. Virol. 30:277-285. 14. Nodenfeld, E., and L. Kjellen. 1975. Dane particles, DNA polymerase, and e-antigen in two different categories of hepatitis B antigen carriers. Intervirology 5:225-232. 15. Okada, K., I. Kamiyama, M. Inomata, M. Imai, Y. Miyakawa, and M. Mayumi. 1976. e antigen and antie 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. 16. Overby, L. R., P. P. Hung, J. C-H. Mao, and C. M. Ling. 1975. Rolling circular DNA associated with Dane particles in hepatitis B virus. Nature (London) 255:84-85. 17. Robinson, W. S., D. A. Clayton, and R. L. Greenman. 1974. DNA of a human hepatitis B virus candidate. J. Virol. 14:384-391. 18. Tsuda, F., K. Takahashi, T. Takahashi, Y. Miyakawa, and M. Mayumi. 1975. Determination of antibody to hepatitis B core antigen by means of immune adherence hemagglutination. J. Immunol. 115:834-838.

anti-e system.

INFECTION AND IMMUNITY, Sept. 1976, Vol. 14, No. 3 p. 631-635 Printed in U.S.A. Copyright C 1976 American Society for Microbiology Hepatitis B An...
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