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J. gen. ViroL (1979), 42, 645-649 Printed in Great Britain
Association of Hepatitis B e-Antigen (HBeAg) Determinants with the Core of Dane Particles ( A c c e p t e d I N o v e m b e r I978 ) SUMMARY
Immunoprecipitates obtained by reacting Dane particle cores with human antibodies to hepatitis B core antigen (HBcAg) were chromatographed on columns of Sepharose 4 B CL using 3 M-NaSCN as eluant. An antigen having the size and immunological specificity of monomeric e-antigen (HBeAg) was separated from HBcAg by this method. Antisera from animals immunized with HBcAg or HBeAg reacted not only with the antigen used for immunization but also with HBeAg and HBcAg, respectively. This indicates that HBeAg determinants are associated with the core of Dane particles. The detection of hepatitis B e-antigen (HBeAg) in sera is correlated with the presence of Dane particles (Nordenfelt & Kjell6n, I975; Takahashi et al. I976), with infectivity of the sera (Alter et al. 1976; Okada et al. 1976; Beasley et al. I977; Shikata et al. I977) and with the detection of hepatitis B core antigen (HBcAg) in liver nuclei (Murphy et al. I976; Trepo et al. I976). Although the antigenic determinants of HBcAg and HBeAg can be distinguished on the basis of reactions with human antibodies (anti-HBc and anti-HBe, respectively (Takahashi et al. 1976; Neurath et al. I978 c)) the following preliminary findings suggest that HBeAg determinants may be associated with the nucleoprotein core of Dane particles: (a) a Rhesus monkey antiserum to HBcAg, purified from hepatitis B virus (HBV)-infected livers (antiserum V8o5-5o 1-563 supplied by the Research Resources Branch of the National Institute of Allergy and Infectious Diseases, Bethesda, Md.), was positive for anti-HBe (J. Vnek, personal communication) in a radioimmunoassay (RIA) test (Mushahwar et al. I978); (b) apparently monomeric HBeAg is similar to HBcAg with respect to isoelectric point and contains a polypeptide having the mol. wt. of the major component of HBcAg (Neurath et al. I978a, c); (e) purified HBcAg (Neurath et al. I978a) after reduction and alkylation, carried out as described (Neurath et al. I978c), followed by treatment with ethanol (final concentration 5o to 6o %, v/v; Budkowska, I977 ) lost its reactivity with anti-HBc in agreement with results described by Budkowska (1977) but was positive for HBeAg as determined by the respective RIA tests (Neurath et al. 1978 b, c). Results presented here provide further evidence for the physical association of HBeAg determinants with HBcAg. Although human anti-HBc IgG isolated from sera containing HBeAg reacted in RIA tests only with HBcAg but not with HBeAg (Neurath et al. I978c), the Rhesus monkey antiserum mentioned before contained both anti-HBc and antibodies to the apparently free and monomeric HBeAg described earlier (Neurath et al. I978 e; Table i). The presence of anti-HBe in the antiserum could be due either to the presence of HBeAg-determinants on HBcAg used for immunization or due to contamination of the HBcAg preparation with HBeAg. To distinguish between these two possibilities, rabbit antisera to HBeAg were prepared and their reaction with monomeric HBeAg and with HBcAg was followed by RIA tests. Human anti-HBe isolated from sera of hepatitis B surface antigen (HBsAg) oo22-I317/79/oooo-3454 $o2.oo~ I979 SGM
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Table I. A n t i - H B c and anti-HBe antibodies in human H B s A g carriers and in animals immunized with Dane particle cores or with H B e A g Characterization of serum Human anti-HBc Human anti-HBc (from HBeAg-positive serum) Rhesus monkey anti-HBc~ Rabbit anti-HBe§ Rabbit anti-HBell Rabbit anti-HBc¶
Anti-HBc titre* I : 5000 I : 5000 I : 500o I : I6 I : 12 r :800
Anti-HBe titret I : 160o < I :2 I : 320 x : tz8 1:24 I :I6
* Corresponding to 50 ~ inhibition end-points (Neurath et al. w78b). Dane particle cores used in the test -- IO #1 of fraction No. I4 (Fig. I). t Corresponding to 50 ~ inhibition end-points. Apparently monomeric HBeAg (Neurath et al. r978c) was used in the test. The relationship between anti-HBc or anti-HBe titres and the absolute concentration of the respective antibodies is not known. Immunizing antigen = Dane particle cores isolated from infected livers. § Prepared as described by Neurath et al. (I976). The antigen used for immunization consisted predominantly of HBeAg bound to IgG. II The antigen used for immunization was apparently monomeric HBeAg free of HBcAg and HBsAg determinants as determined by RIA tests. ¶ The antigen used for immunization was HBcAg corresponding to fractions I4+ I5 (Fig. I). carriers could not be used for this purpose since these sera also always contain antibodies to HBcAg determinants. Results shown in Table I indicate that the rabbit anti-HBe sera were positive in an R I A inhibition test for anti-HBc. This suggests that HBeAg determinants are exposed on a considerable portion of HBcAg particles. More direct evidence for the physical association of HBeAg determinants with HBcAg is based on the following experiment. Dane particles were sedimented from about 3oo ml of HBeAg-positive serum (which had been clarified by centrifugation at roooo rev/min for 2o rain) at 25ooo rev/min for 5 h in the SW 25"2 rotor (Beckman Instruments, Palo Alto, California). The pellet was resuspended in Io ml of 'disruption buffer' (o-t M-NH4CIo'o8 M-MgC12-o'25 ~ mercaptoethanol-o.o5 ~ Nonidet P4o (NP4o), p H 7"6; Neurath et al. I978a) and incubated for 3o min at 37 °C in order to release HBcAg from Dane particles. The preparation was dialysed overnight against o-o I M-tris (hydroxymethyl) aminomethaneo ' I 4 M - N a C I - o ' o 2 ~ NaN3 (TS) containing o.I ~ NP4o. Anti-HBc I g G (2. 5 ml; titre determined by R I A = I:xooo; Neurath et al. I978b) isolated from HBeAg-positive serum and free of anti-HBe detectable by R I A was added to precipitate HBcAg. The mixture was incubated for 3o min at 37 °C, overnight at 4 °C, layered on top of io ml of I5 ~ , v/v, glycerol and centrifuged at 25ooo rev/min for I h in the SW 25.I rotor. The pellet was suspended in 2 ml of TS, layered on top of 2 ml of 15 ~ glycerol and centrifuged at 25 ooo rev/min in the SW 65 rotor for I h. The final pellet was dissolved in 3 M-NaSCN and chromatographed by gel filtration on a column (I.5 × 3o cm) of Sepharose 4B CL (Pharmacia, Piscataway, New Jersey) as described in Fig. I. Fractions after gel filtration and the supernantant from the last centrifugation were analysed by R I A for HBcAg, HBeAg and HBsAg. The relative concentrations of each of these antigens were determined from R I A calibration curves. To distinguish HBeAg from HBcAg in R I A tests, duplicate samples, diluted with either normal human I g G or with anti-HBc I g G (isolated from HBeAgpositive serum), were tested. Similar ct/min indicated the presence of HBeAg, while suppression of ct/min to background levels by anti-HBc indicated the presence of HBcAg (Neurath et al. I978 c). The first peak of radioactivity representing the RIA-positive material
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Fig. I. Gel filtration on a column of Sepharose 4B CL of an HBcAg-anti-HBc immune complex. Arrows correspond to the column void volume 0), to the peak positions of HBsAg [2; tool. wt. 2"5 to 3'7 x io n (Howard & Burrell, 1976)], apoferritin (3; mol. wt. 4"5 x I@), and to the included volume of the column (4); 3 M-NaSCN was used as eluant and one ml fractions were collected. Influenza virus and phenol red were used as markers for the void and included volumes of the column, respectively. Samples (5o #l) of fractions dialysed against TS containing 3 #g/ml of phenylmethy!sulphonylfluoride were tested by RIA, measuring both HBeAg and HBcAg (Neurath et aL I978c). Radioactivity corresponding to negative controls (25 ct/min) was subtracted from the experimental results. eluted just behind the void volume of the column corresponds to HBcAg for the following reasons: (a) its elution pattern is consistent with a mol. wt. of about 9 × Io8 established for Dane particle cores by gel filtration (Fields et al. I977); (b) addition of Ioo #1 of human anti-HBc to fractions corresponding to this peak resulted in negative R I A tests; (c) positive R I A results were obtained when beads coated with anti-HBc I g G (isolated from HBeAgpositive sera) instead of I g G containing both anti-HBc and anti-HBe (Neurath et al. I978 c) were used; (d) only trace amounts of HBsAg were detected in the fractions, indicating nearly complete removal of the Dane particle envelope from the nucleoprotein core. The second peak of radioactivity corresponds to HBeAg since: (a) re-chromatography of pooled fractions 3I to 37 (concentrated to I ml) on Sephadex G - I o o using 3 M-NaSCN as eluant revealed that the RIA-positive material had a mol. wt. of approx. 35ooo, established earlier for apparently monomeric HBeAg (Neurath et al. I978 c); (b) addition of human anti-HBc to fractions corresponding to this peak failed to cause any inhibition in R I A tests but addition of h u m a n I g G containing both anti-HBc and anti-HBe resulted in negative R I A tests; (c) R I A tests were negative with beads coated with anti-HBc only; (d) addition of rabbit anti-HBe to the fractions resulted in negative R I A tests. The concentration of HBeAg in the re-dissolved immunoprecipitate used for chromatography was about 5o times higher than in the last supernatant after sedimenting the H B c A g anti-HBc complex, while the level of HBsAg in the supernatant was approx. Io times higher than in the combined fractions after gel-filtration. Therefore, HBeAg recovered from the HBcAg-anti-HBc immunoprecipitate cannot represent a contaminant from 'soluble' HBeAg present in the original serum.
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The co-precipitation of HBeAg determinants with the nucleoprotein core of Dane particles by anti-HBc (free of anti-HBe) and the finding that HBcAg exposed to 3 M-NaSCN reacted with animal anti-HBe and elicited an immune response to HBeAg (Table I) suggests that HBeAg is a component of Dane particle cores released from the cores under appropriate conditions. It remains unknown whether HBcAg and HBeAg determinants are located on distinct proteins, or whether they represent antigenic determinants on the same protein in two distinct conformations as suggested (Neurath et al. I978 c). Lam et al. (1977) concluded that HBeAg was released from Dane particle-rich preparations treated with detergents. However, their experiments failed to distinguish between HBcAg and HBeAg determinants. The interaction of Dane particles with anti-HBe observed by us (Neurath et al. 1976) but not confirmed by others (Gerin et al. I978 ; Takahashi et al. 1978) may be at least partly ascribed to exposure of HBeAg determinants on partially damaged Dane particles (J. Vnek, personal communication) in analogy with exposed HBcAg determinants on such particles (Purcell et al. I974). An association between HBeAg and HBcAg seems to be supported by the localization of these antigens in nuclei of HBV-infected livers (Arnold et al. I977). However, antigenic sites reacting with antibodies directed against both HBcAg and HBeAg were detected at the membrane of infected hepatocytes in cases of acute lobular hepatitis and chronic active hepatitis (Huang & Neurath, I978), suggesting that internal HBV components may be localized on membranes of infected cells in analogy with other viruses (Tung et aL 1976; Biddison et at. 1977; Braciale, I977; Ledbetter et al. 1977; Virelizier et al. I977). If indeed HBeAg may become exposed on the surface of HBV-infected hepatocytes, an immune response to HBeAg may play an active role in modulating the production of Dane particle cores and in eliminating cells involved in their biosynthesis. Such mechanisms would offer a reasonable explanation for the negative correlation between the presence of anti-HBe in serum and the synthesis of HBcAg and HBV. This study was supported by grant HLo9ol 1-15 from the National Heart, Lung and Blood Institute. We thank Drs P. Beasley and C. E. Stevens for HBeAg-positive sera. A. R. NEURATH N. STRICK
The Lindsley F. Kimball Research Institute o f The N e w York Blood Center N e w York, N . Y . 1o021 U.S.A. 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. & ZIMMERMAN,J. H. (1976). T y p e B hepatitis: the infectivity of blood positive for e antigen a n d D N A polymerase after accidental needlestick exposure. New England Journal of Medicine 295, 9o9-913. ARNOLD, W., NIELSEN, J. O., HARDT, F. & MEYER ZUM BUSCHENFELDE, K. J. (1977). Localisation of e-antigen in nuclei of hepatocytes in HBsAg-positive liver diseases. Gut I9, 994-996. BEASLEY, R. P., TREPO, C., STEVENS, C. E. & SZMUNESS, W. (I977). T h e e antigen a n d vertical t r a n s m i s s i o n of hepatitis B surface antigen. American Journal ofEpidemiology xos, 94-98. BIDDISON, W. E., DOHERTY, P. C. & WEBSTER, R. G. (I977). A n t i b o d y to influenza virus matrix protein detects a c o m m o n antigen on the surface of cells infected with T y p e A influenza viruses. Journal of Experimental Medicine x46 , 69o-697. BRACIALE, T.J. (I977). I m m u n o l o g i c recognition of influenza virus-infected cells. II. Expression o f influenza A m a t r i x protein on the infected cell surface a n d its role in recognition by cross-reactive cytotoxic T cells. Journal of Experimental Medicine x46, 673-689BUDKOWSKA, A. (1977). I m m u n o c h e m i c a l a n d morphological studies o f hepatitis B core antigen isolated f r o m the nuclei of hepatocytes. Journal of Infectious Diseases i3~ , 463-467.
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FIELDS, H . A . , HOLLINGER, F. B., DESMYTER, J., MELNICK, J. L. & DREESMAN, G . R . (I977). Biochemical a n d
biophysical properties of hepatitis B core particles derived from Dane particles and infected hepatocytes.
Intervirology 8, 336-350. ~ERIN, J. r., SHIH, J. W.-K., McAULlrrr, V. J. & PURCELL, R. H. 0978). Antigens of hepatitis B virus: failure to detect HBeAg on the surfaces of HBsAg forms. Journal of General Virology 38, 561-566. HOWARD, C . R . & BURRELL, C . J . (I976). Structure and nature of hepatitis B antigen. Progress in Medical Virology 22, 36-IO3. HUAN~, S. N. & NEURATH,A. R. (I978). Immunohistologic demonstration of hepatitis B viral antigens in liver with reference to its significance in liver injury. Laboratory Investigation (in the press). LAM, K. C., TONG, M. S. & RAKELA,J. (I977). Release of e antigen from a Dane particle-rich preparation of hepatitis B virus. Infection and Immunity x6, 403-404. LEDBETTER, J., NOWINSKI,R. C. & EMERY,S. (I977). Viral proteins expressed on the surface of murine leukemia cells. Journal of Virology 22, 65-73. MURPHYn S. L., PETERSON, J. M., SMITH, J. L., GITNICK, G. L.~ AUSLANDER, G. O., BERQUIST, V. R., MAYNARD, D. E.
& PURCELL,R. H. (1976). Correlation between fluorescent antibody detection of hepatitis B core antigen in liver biopsies and the presence of e-antigen in the serum. Injection and Immunity x3, 296-297. MUSHAHWA~,I. K., OVERBY,L. R., EROSNER,G., DEINHARDT, E. & LING, C.-M. (I978). Prevalence of hepatitis B e antigen and its antibody as detected by radioimmunoassays. Journal of Medical Virology 2, 77-87. NEURATH,A. R., TREPO, C., ellEN, M. & PRINCE,A. M. (I976). Identification of additional antigenic sites on Dane particles and the tubular forms of hepatitis B surface antigen. Journal of General Virology 30, 277-285. NEURATH, A. R., HUANG, S.-N. & STRICK, N. (I978a). Some properties of hepatitis B core antigen isolated from serum of infected humans. Journal of General Virology 39, 9I-IOI. NEURATH, A. R., SZMUNESS, W., STEVENS, C. E., STRICK, N. & HARLEY, E. J. (1978 b). Radioimmunoassay and some properties of human antibodies to hepatitis B core antigen. Journal of General Virology 38, 549-559. NEURATH, A. R., STRICK, N., SZMUNESS, W., STEVENS, C.E. & HARLEY, E.J. ( I 9 7 8 C ) . Radioimmunoassay of hepatitis B e-antigen (HBeAg): identification of HBeAg not associated with immunoglobulins. Journal of General Virology 42 (in the press). NORDENFELT, E. & KJELL~N, L. (I975). Dane particles, D N A polymerase, and e-antigen in two different categories of hepatitis B antigen carriers. Intervirology 5, 225-232. OKADA, K., KAMIYAMA, I., tNOMATA, M., IMAI, M., MIYAKAWA, Y. & MAYUMI, M. (I976). 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. New England Journal of Medicine 294, 746-749. PURCELL, a. H., GERIN, J. L., ALMEIDA,J. B. & HOLLAND,V. V. (I974). Radioimmunoassay for the detection of the core of the Dane particle and antibody to it. Intervirology 2, 231-243. SHIKATA, T., KARASAWA, T., ABE, K., UZAWA, T., SUZUKI, H., ODA, T., IMAI, M., MAYUMI, M. & MORITSUGU, Y.
(I977). Hepatitis B e antigen and infectivity of hepatitis B virus. Journal of Infectious Diseases x36, 571-576. TAKAHASHI, K., IMAI,M., TSUDA,F., TAKAHASHI,T., MIYAKAWA,Y. & MAYUMI,M. (1976). Association of Dane particles with e antigen in the serum of asymptomatic carriers of hepatitis B surface antigen. Journal of Immunology xi7, lO2-1o5. TAKAHASHI, K. YAMASHITA, S., IMAt, M., MIYAKAWA, Y. & MAYUMI, M. (1978). F a i l u r e o f a n t i b o d y t o e antigen to precipitate Dane particles containing D N A polymerase activity and hepatitis B core antigen. Journal of General Virology 38, 431-436. TREPO, C. G., MAGNIUS, L. O., SCHAEFER, R. A. & PRINCE, A. M. (1976). Detection of e antigen and antibody. Correlations with hepatitis B surface and hepatitis B core antigens, liver disease, and outcome of hepatitis B infections. Gastroenterology 7 x, 8o4-8o8. TUNG, J.-S., YOSHIKI, T. & FLEISSNER,E. (I976). A core polyprotein of murine leukemia virus on the surface of mouse leukemia cells. Cell 9, 573-578. VIRELIZIER, J. L., ALLISON, A. C., OXFORD, J. S. & SCHILD, G. C. (I977). Early presence of ribonucleoprotein antigen on surface of influenza virus-infected cells. Nature, London 266, 52-54.
(Received I September I978)
J. gen. ViroL (1979), 42, 645-649 Printed in Great Britain
Association of Hepatitis B e-Antigen (HBeAg) Determinants with the Core of Dane Particles ( A c c e p t e d I N o v e m b e r I978 ) SUMMARY
Immunoprecipitates obtained by reacting Dane particle cores with human antibodies to hepatitis B core antigen (HBcAg) were chromatographed on columns of Sepharose 4 B CL using 3 M-NaSCN as eluant. An antigen having the size and immunological specificity of monomeric e-antigen (HBeAg) was separated from HBcAg by this method. Antisera from animals immunized with HBcAg or HBeAg reacted not only with the antigen used for immunization but also with HBeAg and HBcAg, respectively. This indicates that HBeAg determinants are associated with the core of Dane particles. The detection of hepatitis B e-antigen (HBeAg) in sera is correlated with the presence of Dane particles (Nordenfelt & Kjell6n, I975; Takahashi et al. I976), with infectivity of the sera (Alter et al. 1976; Okada et al. 1976; Beasley et al. I977; Shikata et al. I977) and with the detection of hepatitis B core antigen (HBcAg) in liver nuclei (Murphy et al. I976; Trepo et al. I976). Although the antigenic determinants of HBcAg and HBeAg can be distinguished on the basis of reactions with human antibodies (anti-HBc and anti-HBe, respectively (Takahashi et al. 1976; Neurath et al. I978 c)) the following preliminary findings suggest that HBeAg determinants may be associated with the nucleoprotein core of Dane particles: (a) a Rhesus monkey antiserum to HBcAg, purified from hepatitis B virus (HBV)-infected livers (antiserum V8o5-5o 1-563 supplied by the Research Resources Branch of the National Institute of Allergy and Infectious Diseases, Bethesda, Md.), was positive for anti-HBe (J. Vnek, personal communication) in a radioimmunoassay (RIA) test (Mushahwar et al. I978); (b) apparently monomeric HBeAg is similar to HBcAg with respect to isoelectric point and contains a polypeptide having the mol. wt. of the major component of HBcAg (Neurath et al. I978a, c); (e) purified HBcAg (Neurath et al. I978a) after reduction and alkylation, carried out as described (Neurath et al. I978c), followed by treatment with ethanol (final concentration 5o to 6o %, v/v; Budkowska, I977 ) lost its reactivity with anti-HBc in agreement with results described by Budkowska (1977) but was positive for HBeAg as determined by the respective RIA tests (Neurath et al. 1978 b, c). Results presented here provide further evidence for the physical association of HBeAg determinants with HBcAg. Although human anti-HBc IgG isolated from sera containing HBeAg reacted in RIA tests only with HBcAg but not with HBeAg (Neurath et al. I978c), the Rhesus monkey antiserum mentioned before contained both anti-HBc and antibodies to the apparently free and monomeric HBeAg described earlier (Neurath et al. I978 e; Table i). The presence of anti-HBe in the antiserum could be due either to the presence of HBeAg-determinants on HBcAg used for immunization or due to contamination of the HBcAg preparation with HBeAg. To distinguish between these two possibilities, rabbit antisera to HBeAg were prepared and their reaction with monomeric HBeAg and with HBcAg was followed by RIA tests. Human anti-HBe isolated from sera of hepatitis B surface antigen (HBsAg) oo22-I317/79/oooo-3454 $o2.oo~ I979 SGM
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Table I. A n t i - H B c and anti-HBe antibodies in human H B s A g carriers and in animals immunized with Dane particle cores or with H B e A g Characterization of serum Human anti-HBc Human anti-HBc (from HBeAg-positive serum) Rhesus monkey anti-HBc~ Rabbit anti-HBe§ Rabbit anti-HBell Rabbit anti-HBc¶
Anti-HBc titre* I : 5000 I : 5000 I : 500o I : I6 I : 12 r :800
Anti-HBe titret I : 160o < I :2 I : 320 x : tz8 1:24 I :I6
* Corresponding to 50 ~ inhibition end-points (Neurath et al. w78b). Dane particle cores used in the test -- IO #1 of fraction No. I4 (Fig. I). t Corresponding to 50 ~ inhibition end-points. Apparently monomeric HBeAg (Neurath et al. r978c) was used in the test. The relationship between anti-HBc or anti-HBe titres and the absolute concentration of the respective antibodies is not known. Immunizing antigen = Dane particle cores isolated from infected livers. § Prepared as described by Neurath et al. (I976). The antigen used for immunization consisted predominantly of HBeAg bound to IgG. II The antigen used for immunization was apparently monomeric HBeAg free of HBcAg and HBsAg determinants as determined by RIA tests. ¶ The antigen used for immunization was HBcAg corresponding to fractions I4+ I5 (Fig. I). carriers could not be used for this purpose since these sera also always contain antibodies to HBcAg determinants. Results shown in Table I indicate that the rabbit anti-HBe sera were positive in an R I A inhibition test for anti-HBc. This suggests that HBeAg determinants are exposed on a considerable portion of HBcAg particles. More direct evidence for the physical association of HBeAg determinants with HBcAg is based on the following experiment. Dane particles were sedimented from about 3oo ml of HBeAg-positive serum (which had been clarified by centrifugation at roooo rev/min for 2o rain) at 25ooo rev/min for 5 h in the SW 25"2 rotor (Beckman Instruments, Palo Alto, California). The pellet was resuspended in Io ml of 'disruption buffer' (o-t M-NH4CIo'o8 M-MgC12-o'25 ~ mercaptoethanol-o.o5 ~ Nonidet P4o (NP4o), p H 7"6; Neurath et al. I978a) and incubated for 3o min at 37 °C in order to release HBcAg from Dane particles. The preparation was dialysed overnight against o-o I M-tris (hydroxymethyl) aminomethaneo ' I 4 M - N a C I - o ' o 2 ~ NaN3 (TS) containing o.I ~ NP4o. Anti-HBc I g G (2. 5 ml; titre determined by R I A = I:xooo; Neurath et al. I978b) isolated from HBeAg-positive serum and free of anti-HBe detectable by R I A was added to precipitate HBcAg. The mixture was incubated for 3o min at 37 °C, overnight at 4 °C, layered on top of io ml of I5 ~ , v/v, glycerol and centrifuged at 25ooo rev/min for I h in the SW 25.I rotor. The pellet was suspended in 2 ml of TS, layered on top of 2 ml of 15 ~ glycerol and centrifuged at 25 ooo rev/min in the SW 65 rotor for I h. The final pellet was dissolved in 3 M-NaSCN and chromatographed by gel filtration on a column (I.5 × 3o cm) of Sepharose 4B CL (Pharmacia, Piscataway, New Jersey) as described in Fig. I. Fractions after gel filtration and the supernantant from the last centrifugation were analysed by R I A for HBcAg, HBeAg and HBsAg. The relative concentrations of each of these antigens were determined from R I A calibration curves. To distinguish HBeAg from HBcAg in R I A tests, duplicate samples, diluted with either normal human I g G or with anti-HBc I g G (isolated from HBeAgpositive serum), were tested. Similar ct/min indicated the presence of HBeAg, while suppression of ct/min to background levels by anti-HBc indicated the presence of HBcAg (Neurath et al. I978 c). The first peak of radioactivity representing the RIA-positive material
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Fig. I. Gel filtration on a column of Sepharose 4B CL of an HBcAg-anti-HBc immune complex. Arrows correspond to the column void volume 0), to the peak positions of HBsAg [2; tool. wt. 2"5 to 3'7 x io n (Howard & Burrell, 1976)], apoferritin (3; mol. wt. 4"5 x I@), and to the included volume of the column (4); 3 M-NaSCN was used as eluant and one ml fractions were collected. Influenza virus and phenol red were used as markers for the void and included volumes of the column, respectively. Samples (5o #l) of fractions dialysed against TS containing 3 #g/ml of phenylmethy!sulphonylfluoride were tested by RIA, measuring both HBeAg and HBcAg (Neurath et aL I978c). Radioactivity corresponding to negative controls (25 ct/min) was subtracted from the experimental results. eluted just behind the void volume of the column corresponds to HBcAg for the following reasons: (a) its elution pattern is consistent with a mol. wt. of about 9 × Io8 established for Dane particle cores by gel filtration (Fields et al. I977); (b) addition of Ioo #1 of human anti-HBc to fractions corresponding to this peak resulted in negative R I A tests; (c) positive R I A results were obtained when beads coated with anti-HBc I g G (isolated from HBeAgpositive sera) instead of I g G containing both anti-HBc and anti-HBe (Neurath et al. I978 c) were used; (d) only trace amounts of HBsAg were detected in the fractions, indicating nearly complete removal of the Dane particle envelope from the nucleoprotein core. The second peak of radioactivity corresponds to HBeAg since: (a) re-chromatography of pooled fractions 3I to 37 (concentrated to I ml) on Sephadex G - I o o using 3 M-NaSCN as eluant revealed that the RIA-positive material had a mol. wt. of approx. 35ooo, established earlier for apparently monomeric HBeAg (Neurath et al. I978 c); (b) addition of human anti-HBc to fractions corresponding to this peak failed to cause any inhibition in R I A tests but addition of h u m a n I g G containing both anti-HBc and anti-HBe resulted in negative R I A tests; (c) R I A tests were negative with beads coated with anti-HBc only; (d) addition of rabbit anti-HBe to the fractions resulted in negative R I A tests. The concentration of HBeAg in the re-dissolved immunoprecipitate used for chromatography was about 5o times higher than in the last supernatant after sedimenting the H B c A g anti-HBc complex, while the level of HBsAg in the supernatant was approx. Io times higher than in the combined fractions after gel-filtration. Therefore, HBeAg recovered from the HBcAg-anti-HBc immunoprecipitate cannot represent a contaminant from 'soluble' HBeAg present in the original serum.
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The co-precipitation of HBeAg determinants with the nucleoprotein core of Dane particles by anti-HBc (free of anti-HBe) and the finding that HBcAg exposed to 3 M-NaSCN reacted with animal anti-HBe and elicited an immune response to HBeAg (Table I) suggests that HBeAg is a component of Dane particle cores released from the cores under appropriate conditions. It remains unknown whether HBcAg and HBeAg determinants are located on distinct proteins, or whether they represent antigenic determinants on the same protein in two distinct conformations as suggested (Neurath et al. I978 c). Lam et al. (1977) concluded that HBeAg was released from Dane particle-rich preparations treated with detergents. However, their experiments failed to distinguish between HBcAg and HBeAg determinants. The interaction of Dane particles with anti-HBe observed by us (Neurath et al. 1976) but not confirmed by others (Gerin et al. I978 ; Takahashi et al. 1978) may be at least partly ascribed to exposure of HBeAg determinants on partially damaged Dane particles (J. Vnek, personal communication) in analogy with exposed HBcAg determinants on such particles (Purcell et al. I974). An association between HBeAg and HBcAg seems to be supported by the localization of these antigens in nuclei of HBV-infected livers (Arnold et al. I977). However, antigenic sites reacting with antibodies directed against both HBcAg and HBeAg were detected at the membrane of infected hepatocytes in cases of acute lobular hepatitis and chronic active hepatitis (Huang & Neurath, I978), suggesting that internal HBV components may be localized on membranes of infected cells in analogy with other viruses (Tung et aL 1976; Biddison et at. 1977; Braciale, I977; Ledbetter et al. 1977; Virelizier et al. I977). If indeed HBeAg may become exposed on the surface of HBV-infected hepatocytes, an immune response to HBeAg may play an active role in modulating the production of Dane particle cores and in eliminating cells involved in their biosynthesis. Such mechanisms would offer a reasonable explanation for the negative correlation between the presence of anti-HBe in serum and the synthesis of HBcAg and HBV. This study was supported by grant HLo9ol 1-15 from the National Heart, Lung and Blood Institute. We thank Drs P. Beasley and C. E. Stevens for HBeAg-positive sera. A. R. NEURATH N. STRICK
The Lindsley F. Kimball Research Institute o f The N e w York Blood Center N e w York, N . Y . 1o021 U.S.A. REFERENCES
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(Received I September I978)
