THE JOURNAL OF I:X~'EGnOUS DiSEASES. VOL. 139, 1'\0, 4 • APRiL 19',9 © 1979 by The University of Chicago. 0022-1899179/3904-0004$00.81
A Partial Characterization of Hepatitis B e Antigen From the Divisions of Gastroenterology and Infectious Diseases, Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri
Clinton R. Weil, David L. Hilton, Wanda Wellinghoff, Lawrence D. Gelb, Robert P. Perrillo, and Richard D. Aach
In 1972 Magnius and Espmark (1] identified an immunologically distinct antigen, the e antigen (HBeAg), in the serum of patients with type B hepatitis. Both HBeAg and its antibody, antiHBe, are markers frequently found in the serum of patients with chronic hepatitis B. The presence of HBeAg appears to correlate with the presence of a large number of circulating Dane particles [2] and a high level of DNA polymerase activity [3], whereas the opposite is true of antiHBe [3]. The presence of HBeAg also appears to be related to infectivity [4], and its persistence seems to be related to chronicity [5] of type B infection. It has been suggested by some investigators that HBeAg is either an antigenic component of the Dane particle itself or a soluble protein released in response to infection with hepatitis B virus [5]. Others have proposed that HBeAg is a large
protein that binds hepatitis B surface antigen (HBsAg) to the Dane particle inner core [3]. An early hypothesis that HBeAg represents the DNA polymerase protein [6] has since been refuted by additional investigation [7, 8]. Recently, it has been claimed that HBeAg has the properties of IgG, and it has been suggested that the e-anti-e system may correspond to an idiotype-anti-idiotype system [9]. HBeAg has also been associated with a lactic acid dehydrogenase isoenzyme [lO]. Most recently, Takahashi et al. [II] have shown that HBeAg can exist in a free state, as well as bound to IgG. The nature of HBeAg is unclear; therefore, we initiated the following investigation to characterize HBeAg. Materials and Methods
HBsAg-positive sera (Ausria II,® Abbott Laboratories, North Chicago, Ill.) from asymptomatic carriers were examined for HBeAg and antiHBe by rheophoresis (Rheotect" plates, Abbott) as previously described [2], with use of sera known to contain HBeAg and anti-HBe. The HBeAgpositive sera identified in this fashion were also tested for DNA polymerase activity by the method of Kaplan et al. [12]. Those sera with high levels of activity were chosen for HBeAg preparation. Sera containing anti-HBe were uniformly negative for DNA polymerase. Neither the specimens containing HBeAg nor those contain-
Received for publication March 24, 1978, and in revised form October 26, 1978. This work was supported in part by training grant no. 5T01 AlO0459 from the National Institutes of Health. Dr. Gelb is an investigator of the Howard Hughes Medical Institute. We thank Drs. G. LeBouvier, W. Szmuness, G. Vyas, and S. Potertz for providing reference samples of hepatitis B e antigen and its antibody for standardization and compariSOIl.
Please address requests for reprints to Dr. Clinton R. Weil, c/o Dr. Richard D. Aach, Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
401
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Partially purified hepatitis B e antigen (HBeAg) was prepared by ultracentrifugation, ammonium sulfate precipitation, and molecular sieve chromatography of sera obtained from asymptomatic carriers of hepatitis B surface antigen. The antigenic specificity of the HBeAg preparations was investigated further with affinity chromatography. The results indicated that HBeAg is distinct and separable from DNA polymerase activity. Columns coupled with either goat IgG prepared from antiserum to human IgG or antibody to HBeAg bound all detectable HBeAg and bound 31 % and 100% of the IgG, respectively, from a partially purified HBeAg preparation. Rate zonal sucrose sedimentation and molecular sieve and ion-exchange chromatography indicated a variability in molecular weight and charge; this finding suggested a heterogeneous population of immunoreactivities containing HBeAg. Our preliminary results suggest the existence of an HBeAg-IgG complex.
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PBS 2 • All respective fractions were pooled. Each of the six fractions plus the pellet were assayed for DNA polymerase activity. Fractions 5 and 6, which contained the HBeAg peak, were pooled, and saturated ammonium sulfate was added to a concentration of 20% of the final volume. The centrifuged precipitate was discarded, and ammonium sulfate was added to the supernatant to make a 40% solution with respect to the original volume. The 20%-40% precipitate was resuspended in and dialyzed against PBS). After dialysis, 1.0-ml aliquots were chromatographed on a Sephadex G-200 column (Pharmacia; 1.2 em X 20 em, 25-ml bed volume). PBS 1 was pumped as the eluent through the column at a rate of 4.0 ml rhr. Fractions of 1.0 ml were collected and assayed for HBeAg, for protein [13], and for IgG. IgG was assayed by radial immunodiffusion on agar plates (Meloy Laboratories, Springfield, Va.). Fractions containing the HBeAg peak from each chromatography were pooled and concentrated fivefold by ammonium sulfate precipitation (20%-40% precipitate). Affinity chromatograph». Sepharose 4B activated with cyanogen bromide (Sigma Chemical Co., St. Louis, Mo.) was coupled to appropriate ligands by the method of Axen et al. [14]. Boratebuffered saline, pH 8.0, 0.5 M N aCl, was used as both a coupling buffer and a running buffer. The following ligands were used: goat IgG obtained from antiserum to human IgG (GAHlgG), normal goat IgG, human IgG, human serum containing rheumatoid factor, and human IgG containing anti-HBe (anti-HBe-IgG). The affinity chromatography was carried out batchwise in test tubes, a method which permitted 30-min contact times with the material to be chromatographed and centrifugal washing. The protein content of each fraction before and after absorption was assayed by the method of Lowry et al. [13]. The IgG content of each fraction was also measured before and after absorption by radial immunodiffusion. Alternative purification procedures. Other avenues of approach to the purification of HBeAg were employed. In step I (figure I), instead of the initial ultracentrifugation procedure, clarified HBeAg-positive sera were precipitated with ammonium sulfate. Aliquots of the resuspended 20%-40% precipitate were applied to a Sephadex G-200 column (1.5 em X 100 em, l70-ml bed
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ing anti-HBe were positive for antibody to HBsAg (anti-HBs) (AUSAB,® Abbott), but both groups of specimens contained antibody to hepatitis B core antigen (anti-HBc) (CORAB,® Abbott; a solid-phase radioimmunoassay for core protein that uses an antibody with a 125 1 radiolabel). Sera obtained from healthy individuals and found to be negative for HBeAg, anti-HBs, and anti-HBc served as controls. Purification of IgG containing anti-HBe. A 20.0-ml volume of serum containing anti-HBe was centrifuged at 2,000 g for 10 min for removal of lipids and remaining erythrocytes, filtered through a MiIIipore filter (pore size, 0.45 fJ-m; Millipore Corp., Bedford, Mass.), and treated sequentially with ammonium sulfate. Saturated ammonium sulfate was added to a concentration of 20% by volume, the pellet was removed by centrifugation, and ammonium sulfate was added to the supernatant to bring it to a concentration of 40% ammonium sulfate with respect to the original volume. The second precipitate was suspended in and dialyzed against phosphate-buffered saline (PBS 1) , pH 7.4, 0.7 M NaCl. This 40% precipitate solution was passed through a column of DEAE-Sephadex (Pharmacia Fine Chemicals, Piscataway, N.J.; 1.2 em X 20 em with P0 4 2 - as anion). The fractions immediately subsequent to the void volume were pooled and concentrated with an Amicon concentrator (Amicon Corp., Lexington, Mass.). This resultant preparation of anti-HBe produced a strong precipitin line against HBeAg after ] 2-] 8 hr when assayed on rheophoresis plates. Preparation of HBeAg. A flow chart of methods for the partial purification of HBeAg is shown in figure 1. Specifically" 20.0 ml of serum containing HBeAg was centrifuged at 2,000 g for ]0 min for removal of lipids and remaining erythrocytes, diluted to 60.0 ml with phosphatebuffered saline (PBS 2) , pH 7.4, 0.]4 M NaC!, and filtered through a MiIIipore filter (pore size, :).45 fLm). Aliquots of 5.0 ml were layered onto twelve 5.0-ml, 30% (wt/vol) sucrose (in PBS 1 ) cushions and spun for ] 7 hr at 45,000 rpm in a Spinco 50-Ti rotor (Beckman Instruments, Spinco Division, Palo Alto, Calif.). Six fractions of 1.5 ml were removed sequentially from the top of each tube, and the pellet was resuspended in 2.0 ml of
Weil et al.
Partial Characterization of HBeAg
Flow chart of methods for partial purification of hepatitis B e antigen.
volume). Fractions of 2.0 ml were collected, concentrated with Lyphogel" (Gelman Instrument Co., Ann Arbor, Mich.), and assayed for HBeAg. HBeAg-positive fractions were pooled and applied to a Biegel" A5-M column (Biorad Laboratories, Richmond, Cali£.; 1.5 em X 100 em, 170-ml bed volume). Fractions of 2.0 ml were collected and assayed again for HBeAg. Sucrose cushion. In steps 2 and 3 (figure 1), =larified and filtered HBeAg-positive serum was
layered onto a 30% (wtjvol) sucrose (in PBS 1 ) cushion and spun in a Spinco 50-Ti rotor at 45,000 rpm for 38 hr and 4 hr, respectively. Sucrose (20%-50%) gradient. In step 4, HBeAg-positive fractions 5 and 6 from the l7-hr 30% sucrose cushion were dialyzed against PBS], layered onto a 20%-50% sucrose (in PBS 1 ) gradient, and spun for 4 hr in a Spinco SW-40 rotor at 30,000 rpm. Fractions of 1.0 ml were collected from the bottom by puncture of the
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Figure 1.
40!l
404
Results
Anti-HBe-IgG. The resultant anti-HBeIgG preparation was free from HBsAg, as determined by Ausria II, and contained 85%-95% IgG / mg of protein. Upon rheophoresis, this
preparation regularly formed a strong precipitin line with HBeAg after 12-18 hr. HBeAg. The method described yielded partially purified HBeAg. Table I shows that the initial centrifugation step removed the DNA polymerase activity but not all of the HBsAg. Essentially all of the DNA polymerase activity present in the starting HBeAg-positive sera was recovered in the pellet fraction. Fractions 5 and 6 lacked DNA polymerase activity and contained all of the detectable HBeAg. The density of these fractions (rate zonal sedimentation) was between 1.13 and 1.14 g/cm 3 . HBsAg was found to be widely scattered at densities of > 1.10 g/cm3 • Fractions 5 and 6 from all 12 tubes were pooled (40 ml containing 375 mg of protein after dialysis) and treated with ammonium sulfate (see figure I). The 20%-40% precipitate was resuspended, dialyzed against PBSt, and concentrated to 5.5 ml with Lyphogel. This pool contained 60 mg of protein, of which 48.5 mg was IgG. It was also positive for HBeAg, HBsAg, and antiHBc. At this point in the purification, 4.3% of the total protein and 69% of the IgG had been recovered. An assay for DNA polymerase revealed no activity. Thus, the initial centrifugaTable 1. Results of ultracentrifugation of representative serum samples containing hepatitis B e antigen (HBeAg) on a 30% sucrose cushion.
Fraction * I
'2 3 4 5 6 Pellet Starting serum **
DNA polymerase activity T Density
60 80 75 77
75 94 433 400
1.010 1.018 1.058 1.109 1.131 1.142
+ HBeAg§
+ ++ +
HBsAgl1
1,539 1,360 3,579 8,369 9,702 8,181 6,981 12,931
'Volume of 1.5 ml from the top of a l O-ml sucrose cushion. tAmount of 'H (cpm) incorporated by a I.O-ml sample concentrated by centrifugation to 0.1 m!. tNumber of g/em' of sample from fraction containing sucrose and serum. §The symbols + and - indicate that the sample does or does not form a precipitin line against antibody to HBeAg upon rheophoresis, respectively. + + = maximal immunoprecipitate. II Amount of 125 1 (cpm) in 0.25 ml, as determined by Ausria II radioimmunoassay. HBsAg = hepatitis B surface antigen. "Data for DNA polymerase have been volume corrected for comparison.
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tube and displacement of the fractions with paraffin oil. The fractions were dialyzed individually against PBS t and assayed for HBeAg. A micon partition. In step 5, dialyzed fractions 5 and 6 from a 17-hr, 30% sucrose cushion were subjected to partition in an Amicon concentrator by sequential filtration through PMIO and XM300 membranes (Amicon). Aliquots were recovered after each filtration step and assayed for HBeAg. Biogel A5-M chromatography. In step 6, instead of a Sephadex G-200 column, a Bioge! A5M column was used according to the method previously described. Aliquots of the dialyzed 20%40% ammonium sulfate pellet were applied with PBSt as the running buffer. Fractions of 2.0 ml were collected, concentrated with Lyphogel, and assayed for HBeAg. DEAE-Sephadex chromatography. In step 7, instead of either Sephadex G-200 or Biogel A5M columns, a DEAE-Sephadex column was prepared. The same starting material as that described in step 6 was used, dialyzed extensively against the appropriate buffer, and applied to a column (60-ml bed volume) of Whatman DE52 (Whatman Inc., Clifton, N.].) with dimensions of 2.7 em X 17 em. In the first trial, phosphate was used as the anion on the column with 0.01 M sodium phosphate, pH 7.4, as the eluent. Stepwise elution from 0.1 M to 0.5 M phosphate by 0.1 M increments, followed by additional steps at 1.0 M and 3.0 M phosphate, was carried out. A second trial employed 0.01 M Tris-H'Cl buffer, pH 7.4, with chloride as the column anion. Gradient elution from 0.1 M to 0.5 M NaCI was followed by stepwise elution at 1.0 M and 3.0 M N aCl. At least 50 mg of protein (as determined by the method of Lowry et al. [13]) was applied to the column in each trial. Chromatography on a DEAE-Sephadex column was tried again in step 8 after an intervening separation on Sephadex G-200. Both elution techniques were used as described above.
Wei! et al.
Partial Characterization of HBeAg
405
E
200,000 daltons, as shown both by its dispersion in a 20%-50% sucrose gradient and by its elution pattern during Biogel A5-M column chromatography. As Biogel A5-M is generally capable of resolving proteins
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ium sulfate precipitation was next passed over the column to fill all available nonspecific binding sites even after inactivation with ethanolamine. Approximately 1.5 mg bound. When an HBeAg preparation containing 9.6 mg of protein and 5.85 mg of IgG was passed over the column, all the HBeAg as well as >99% of the total protein and IgG was recovered. Thus, the HBeAg preparation contained no detectable activity against human immunoglobulins. An affinity chromatography column was prepared with anti-HBe as the ligand by coupling 116 mg of anti-HBe-IgG to a lO-ml volume of Sepharose 4B. Protein (31.2 mg) from a 20%-40% ammonium sulfate precipitate of normal human serum was then added to fill all of the available nonspecific binding sites. A sample (20 mg) of the same preparation of normal human serum was separately added to a 2.0-ml aliquot of the Sepharoseanti-HBe conjugate. Of the initial 20 mg, 99.9% was recovered from the pass-through volume and wash, a result indicating that no such nonspecific sites remained available. An HBeAg preparation was added to the remaining coupled Sepharose. As seen in table 2, the Sepharose-anti-HBe conjugate bound an equivalent amount of IgG and total protein as well as all of the detectable HBeAg in the preparation. Affinity chromatography with use of normal human serum containing rheumatoid factor as the ligand produced results similar to those obtained with GAHlgG. The column was preloaded with normal human serum to fill nonspecific binding sites. The rheumatoid factor column bound equivalent amounts of IgG and total protein along with all the HBeAg (data not shown). Several systems were employed in an attempt to decouple the HBeAg from the affinity chromatography columns for further analysis (table 3). All attempts were unsuccessful. As shown, reagents with pH values as low as 2.0 and as high as 10.9 were employed. All were allowed contact for 5-60 min with the respective columns. Separate studies were done to determine how long HBeAg would remain immunologically active in each decouplant. The times selected for each zompound were shorter than that which completely destroyed clearly discernible immunologic activity. After contact, each eluent was immediately titrated to pH 7.4 and dialyzed against
407
Partial Characterization of HBeAg
matography columns in sufficient quantities pre· vented both the further analysis of the IgG moiety and the use of affinity chromatography for further purification. The heterogeneity of charge, variability of weight, and behavior on affinity chromatography columns suggest that HBeAg is not simply IgG with an additional immunoreactive site, but that HBeAg and IgG are associated in a complex. These observations are in accord with the recent report of Takahashi et al. [II], who provided evidence that HBeAg can circulate in serum complexed to IgG or in free form. References 1. Magnius, L. 0., Espmark, J. A. New specificities in Australia antigen positive sera distinct from the LeBouvier determinants. J. Immunol. 109:1017-1021, 1972. 2. Perrillo, R. P., Gelb, L. D., Milligan, W. H. III, Wellinghoff, W., Aach, R. D. Discordant e antigen. DNA polymerase activity, and Dane particle responses in two patients representing an index case-contact case pair with hepatitis B virus infection. J. Infect. Dis. 136:117-121,1977. 3. Gitnick, G. L. [moderator], Goldberg, L. S., Koretz, R., Walsh, J. H. [discussants]. The liver and the antigens of hepatitis B. Ann. Intern. Med. 85:488-496, 1976. 4. Rebhun, R. J., Overby, L. R., Gitnick, G. L. Infectivity of asymptomatic e antigen positive and negative HBsAg carriers. Gastroenterology 71:925, 1976. 5. 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. 6. Neurath, A. R., Strick, N. Evidence against the postulated identity of e-antigen with DNA polymerase associated with the hepatitis B candidate virus. Intervirology 7:356-359, 1976. 7. Takahashi, K., Yamashita, S., Imai, M., Miyakawa, Y., Mayumi, M. Failure of antibody to e antigen to precipitate Dane particles containing DNA polymerase activity and hepatitis B core antigen. J. Gen. Virol. 38:431-436, 1978. 8. Gerin, J. L., Shih, J. W.-K., McAuliffe, V. J., Purcell, R. H. Antigens of hepatitis B virus: failure to detect HBeAg on the surfaces of HBsAg forms. J. Gen. Virol. 38:561-566, 1978. 9. Neurath, A. R., Strick, N. Host specificity of a serum marker for hepatitis B: evidence that "e antigen" has the properties of an immunoglobulin. Proc. Natl. Acad. Sci. U.S.A. 74:1702-1706,1977. 10. Vyas, G. N., Peterson, D. L., Townsend, R. M., Damle, S. R., Magnius, L. O. Hepatitis B "e" antigen: an
Downloaded from http://jid.oxfordjournals.org/ at Indiana University Library on July 26, 2015
weighing between 2 X 105 and 106 daltons [18], dispersion would result if a heterogeneous family of immunologically reactive molecules was involved. Rechromatography of the HBeAg peak on Sephadex G-200 demonstrated that it coelutes with the IgG immunoreactive material, which was but a small fraction of the total IgG present in the HBeAg preparation at that stage of the purification. Similarly, a GAHIgG-coupled affinity chromatography column bound both HBeAg and IgG, but not IgM. When rheumatoid factor was used as a ligand on an affinity chromatography column, 19G and HBeAg were bound. It should be noted that the preparations of HBeAg and antiHBe were themselves negative for rheumatoid factor. HBeAg appears not to be a monomeric IgG molecule by itself, since the immunoreactive HBeAg is larger than the IgG peak by column chromatography (figure 2). Likewise, HBeAg does not appear to be a multimeric IgG molecule since such molecules would appear in distinct peaks during Biogel A5-M chromatography. The data obtained by affinity chromatography suggest that there is a specific association between the two immunoreactivities (IgG and HBeAg), rather than that HBeAg has sufficiently similar biophysical and biochemical properties to copurify with normal human IgG. The heterogeneous nature of this association delineated by our studies corroborates the work of Neurath and Strick [9], who have reported that the HBeAg immunoreactivity of their preparations was spread over a pH range of 4.9-7.5 during isoelectric focusing. This observation implies a group of relatively negatively charged molecules and could explain our inability to elute HBeAg from DEAE-Sephadex despite the addition of up to a 3 M concentration of anion. The IgG associated with HBeAg could represent a specific class or subclass of IgG molecules, but no data are available to support.rhis hypothesis. IgG was probably retarded by the anti-HBe-coupled affinity chromatography column because it is presznt in a complex bound to HBeAg. HBeAg and IgG moieties lacking immunoreactivity could be removed from the affinity chromatography colLimns, as measured by protein content, only by highly chaotropic agents. The inability to de· couple the bound HBeAg from the affinity chro-
Weil et al.
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ll.
12.
13.
15.
16.
17. 18.
of peptides and proteins to polysaccharides by means of cyanogen halides. Nature 214: 1302-1304,1967. Neurath, A. R., Trepo, C., Chen, M., Prince, A. M. Identification of additional antigenic sites on Dane particles and the tubular forms of hepatitis B surface antigen. J. Gen. ViroI. 30:277-285, 1976. Takahashi, K., Fukuda, M., Baba, K., Imai, M., Miyakawa, Y., Mayumi, M. Determination of e antigen and antibody to e by means of passive hemagglutination method. J. ImmunoI. 119:1556-1559, 1977. Pharmacia Fine Chemicals Catalogue, 1974. Pharmacia, Piscataway, N.J., 1974, p. 6-7. Tipton, C. L., Paulis, J. W., Pierson, M. D. Gel filtration of lipid mixtures. J. Chromatogr. 14:486-489, 1964.
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14.
apparent association with lactate dehydrogenase isozyme-5. Science 198:1068-1070, 1977. Takahashi, K., Imai, M., Miyakawa, Y., Iwakiri, S., Mayumi, M. Duality of hepatitis B e antigen in serum of persons infected with hepatitis B virus: evidence for the nonidentity of e antigen with immunoglobulins. Proc. NatI. Acad. Sci. U.S.A. 75:1952-1956, 1978. Kaplan, P. M., Greenman, R. L., Gerin, J. L., Purcell, R. H., Robinson, W. S. DNA polymerase associated with human hepatitis B antigen. J. ViroI. 12:9951005, 1973. Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. Protein measurement with the Folin phenol reagent. J. BioI. Chern. 193:265-275, 1951. Axen, R., Porath, J., Ernback, S. Chemical coupling-
A Partial Characterization of Hepatitis B e Antigen From the Divisions of Gastroenterology and Infectious Diseases, Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri
Clinton R. Weil, David L. Hilton, Wanda Wellinghoff, Lawrence D. Gelb, Robert P. Perrillo, and Richard D. Aach
In 1972 Magnius and Espmark (1] identified an immunologically distinct antigen, the e antigen (HBeAg), in the serum of patients with type B hepatitis. Both HBeAg and its antibody, antiHBe, are markers frequently found in the serum of patients with chronic hepatitis B. The presence of HBeAg appears to correlate with the presence of a large number of circulating Dane particles [2] and a high level of DNA polymerase activity [3], whereas the opposite is true of antiHBe [3]. The presence of HBeAg also appears to be related to infectivity [4], and its persistence seems to be related to chronicity [5] of type B infection. It has been suggested by some investigators that HBeAg is either an antigenic component of the Dane particle itself or a soluble protein released in response to infection with hepatitis B virus [5]. Others have proposed that HBeAg is a large
protein that binds hepatitis B surface antigen (HBsAg) to the Dane particle inner core [3]. An early hypothesis that HBeAg represents the DNA polymerase protein [6] has since been refuted by additional investigation [7, 8]. Recently, it has been claimed that HBeAg has the properties of IgG, and it has been suggested that the e-anti-e system may correspond to an idiotype-anti-idiotype system [9]. HBeAg has also been associated with a lactic acid dehydrogenase isoenzyme [lO]. Most recently, Takahashi et al. [II] have shown that HBeAg can exist in a free state, as well as bound to IgG. The nature of HBeAg is unclear; therefore, we initiated the following investigation to characterize HBeAg. Materials and Methods
HBsAg-positive sera (Ausria II,® Abbott Laboratories, North Chicago, Ill.) from asymptomatic carriers were examined for HBeAg and antiHBe by rheophoresis (Rheotect" plates, Abbott) as previously described [2], with use of sera known to contain HBeAg and anti-HBe. The HBeAgpositive sera identified in this fashion were also tested for DNA polymerase activity by the method of Kaplan et al. [12]. Those sera with high levels of activity were chosen for HBeAg preparation. Sera containing anti-HBe were uniformly negative for DNA polymerase. Neither the specimens containing HBeAg nor those contain-
Received for publication March 24, 1978, and in revised form October 26, 1978. This work was supported in part by training grant no. 5T01 AlO0459 from the National Institutes of Health. Dr. Gelb is an investigator of the Howard Hughes Medical Institute. We thank Drs. G. LeBouvier, W. Szmuness, G. Vyas, and S. Potertz for providing reference samples of hepatitis B e antigen and its antibody for standardization and compariSOIl.
Please address requests for reprints to Dr. Clinton R. Weil, c/o Dr. Richard D. Aach, Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
401
Downloaded from http://jid.oxfordjournals.org/ at Indiana University Library on July 26, 2015
Partially purified hepatitis B e antigen (HBeAg) was prepared by ultracentrifugation, ammonium sulfate precipitation, and molecular sieve chromatography of sera obtained from asymptomatic carriers of hepatitis B surface antigen. The antigenic specificity of the HBeAg preparations was investigated further with affinity chromatography. The results indicated that HBeAg is distinct and separable from DNA polymerase activity. Columns coupled with either goat IgG prepared from antiserum to human IgG or antibody to HBeAg bound all detectable HBeAg and bound 31 % and 100% of the IgG, respectively, from a partially purified HBeAg preparation. Rate zonal sucrose sedimentation and molecular sieve and ion-exchange chromatography indicated a variability in molecular weight and charge; this finding suggested a heterogeneous population of immunoreactivities containing HBeAg. Our preliminary results suggest the existence of an HBeAg-IgG complex.
4U2
PBS 2 • All respective fractions were pooled. Each of the six fractions plus the pellet were assayed for DNA polymerase activity. Fractions 5 and 6, which contained the HBeAg peak, were pooled, and saturated ammonium sulfate was added to a concentration of 20% of the final volume. The centrifuged precipitate was discarded, and ammonium sulfate was added to the supernatant to make a 40% solution with respect to the original volume. The 20%-40% precipitate was resuspended in and dialyzed against PBS). After dialysis, 1.0-ml aliquots were chromatographed on a Sephadex G-200 column (Pharmacia; 1.2 em X 20 em, 25-ml bed volume). PBS 1 was pumped as the eluent through the column at a rate of 4.0 ml rhr. Fractions of 1.0 ml were collected and assayed for HBeAg, for protein [13], and for IgG. IgG was assayed by radial immunodiffusion on agar plates (Meloy Laboratories, Springfield, Va.). Fractions containing the HBeAg peak from each chromatography were pooled and concentrated fivefold by ammonium sulfate precipitation (20%-40% precipitate). Affinity chromatograph». Sepharose 4B activated with cyanogen bromide (Sigma Chemical Co., St. Louis, Mo.) was coupled to appropriate ligands by the method of Axen et al. [14]. Boratebuffered saline, pH 8.0, 0.5 M N aCl, was used as both a coupling buffer and a running buffer. The following ligands were used: goat IgG obtained from antiserum to human IgG (GAHlgG), normal goat IgG, human IgG, human serum containing rheumatoid factor, and human IgG containing anti-HBe (anti-HBe-IgG). The affinity chromatography was carried out batchwise in test tubes, a method which permitted 30-min contact times with the material to be chromatographed and centrifugal washing. The protein content of each fraction before and after absorption was assayed by the method of Lowry et al. [13]. The IgG content of each fraction was also measured before and after absorption by radial immunodiffusion. Alternative purification procedures. Other avenues of approach to the purification of HBeAg were employed. In step I (figure I), instead of the initial ultracentrifugation procedure, clarified HBeAg-positive sera were precipitated with ammonium sulfate. Aliquots of the resuspended 20%-40% precipitate were applied to a Sephadex G-200 column (1.5 em X 100 em, l70-ml bed
Downloaded from http://jid.oxfordjournals.org/ at Indiana University Library on July 26, 2015
ing anti-HBe were positive for antibody to HBsAg (anti-HBs) (AUSAB,® Abbott), but both groups of specimens contained antibody to hepatitis B core antigen (anti-HBc) (CORAB,® Abbott; a solid-phase radioimmunoassay for core protein that uses an antibody with a 125 1 radiolabel). Sera obtained from healthy individuals and found to be negative for HBeAg, anti-HBs, and anti-HBc served as controls. Purification of IgG containing anti-HBe. A 20.0-ml volume of serum containing anti-HBe was centrifuged at 2,000 g for 10 min for removal of lipids and remaining erythrocytes, filtered through a MiIIipore filter (pore size, 0.45 fJ-m; Millipore Corp., Bedford, Mass.), and treated sequentially with ammonium sulfate. Saturated ammonium sulfate was added to a concentration of 20% by volume, the pellet was removed by centrifugation, and ammonium sulfate was added to the supernatant to bring it to a concentration of 40% ammonium sulfate with respect to the original volume. The second precipitate was suspended in and dialyzed against phosphate-buffered saline (PBS 1) , pH 7.4, 0.7 M NaCl. This 40% precipitate solution was passed through a column of DEAE-Sephadex (Pharmacia Fine Chemicals, Piscataway, N.J.; 1.2 em X 20 em with P0 4 2 - as anion). The fractions immediately subsequent to the void volume were pooled and concentrated with an Amicon concentrator (Amicon Corp., Lexington, Mass.). This resultant preparation of anti-HBe produced a strong precipitin line against HBeAg after ] 2-] 8 hr when assayed on rheophoresis plates. Preparation of HBeAg. A flow chart of methods for the partial purification of HBeAg is shown in figure 1. Specifically" 20.0 ml of serum containing HBeAg was centrifuged at 2,000 g for ]0 min for removal of lipids and remaining erythrocytes, diluted to 60.0 ml with phosphatebuffered saline (PBS 2) , pH 7.4, 0.]4 M NaC!, and filtered through a MiIIipore filter (pore size, :).45 fLm). Aliquots of 5.0 ml were layered onto twelve 5.0-ml, 30% (wt/vol) sucrose (in PBS 1 ) cushions and spun for ] 7 hr at 45,000 rpm in a Spinco 50-Ti rotor (Beckman Instruments, Spinco Division, Palo Alto, Calif.). Six fractions of 1.5 ml were removed sequentially from the top of each tube, and the pellet was resuspended in 2.0 ml of
Weil et al.
Partial Characterization of HBeAg
Flow chart of methods for partial purification of hepatitis B e antigen.
volume). Fractions of 2.0 ml were collected, concentrated with Lyphogel" (Gelman Instrument Co., Ann Arbor, Mich.), and assayed for HBeAg. HBeAg-positive fractions were pooled and applied to a Biegel" A5-M column (Biorad Laboratories, Richmond, Cali£.; 1.5 em X 100 em, 170-ml bed volume). Fractions of 2.0 ml were collected and assayed again for HBeAg. Sucrose cushion. In steps 2 and 3 (figure 1), =larified and filtered HBeAg-positive serum was
layered onto a 30% (wtjvol) sucrose (in PBS 1 ) cushion and spun in a Spinco 50-Ti rotor at 45,000 rpm for 38 hr and 4 hr, respectively. Sucrose (20%-50%) gradient. In step 4, HBeAg-positive fractions 5 and 6 from the l7-hr 30% sucrose cushion were dialyzed against PBS], layered onto a 20%-50% sucrose (in PBS 1 ) gradient, and spun for 4 hr in a Spinco SW-40 rotor at 30,000 rpm. Fractions of 1.0 ml were collected from the bottom by puncture of the
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Figure 1.
40!l
404
Results
Anti-HBe-IgG. The resultant anti-HBeIgG preparation was free from HBsAg, as determined by Ausria II, and contained 85%-95% IgG / mg of protein. Upon rheophoresis, this
preparation regularly formed a strong precipitin line with HBeAg after 12-18 hr. HBeAg. The method described yielded partially purified HBeAg. Table I shows that the initial centrifugation step removed the DNA polymerase activity but not all of the HBsAg. Essentially all of the DNA polymerase activity present in the starting HBeAg-positive sera was recovered in the pellet fraction. Fractions 5 and 6 lacked DNA polymerase activity and contained all of the detectable HBeAg. The density of these fractions (rate zonal sedimentation) was between 1.13 and 1.14 g/cm 3 . HBsAg was found to be widely scattered at densities of > 1.10 g/cm3 • Fractions 5 and 6 from all 12 tubes were pooled (40 ml containing 375 mg of protein after dialysis) and treated with ammonium sulfate (see figure I). The 20%-40% precipitate was resuspended, dialyzed against PBSt, and concentrated to 5.5 ml with Lyphogel. This pool contained 60 mg of protein, of which 48.5 mg was IgG. It was also positive for HBeAg, HBsAg, and antiHBc. At this point in the purification, 4.3% of the total protein and 69% of the IgG had been recovered. An assay for DNA polymerase revealed no activity. Thus, the initial centrifugaTable 1. Results of ultracentrifugation of representative serum samples containing hepatitis B e antigen (HBeAg) on a 30% sucrose cushion.
Fraction * I
'2 3 4 5 6 Pellet Starting serum **
DNA polymerase activity T Density
60 80 75 77
75 94 433 400
1.010 1.018 1.058 1.109 1.131 1.142
+ HBeAg§
+ ++ +
HBsAgl1
1,539 1,360 3,579 8,369 9,702 8,181 6,981 12,931
'Volume of 1.5 ml from the top of a l O-ml sucrose cushion. tAmount of 'H (cpm) incorporated by a I.O-ml sample concentrated by centrifugation to 0.1 m!. tNumber of g/em' of sample from fraction containing sucrose and serum. §The symbols + and - indicate that the sample does or does not form a precipitin line against antibody to HBeAg upon rheophoresis, respectively. + + = maximal immunoprecipitate. II Amount of 125 1 (cpm) in 0.25 ml, as determined by Ausria II radioimmunoassay. HBsAg = hepatitis B surface antigen. "Data for DNA polymerase have been volume corrected for comparison.
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tube and displacement of the fractions with paraffin oil. The fractions were dialyzed individually against PBS t and assayed for HBeAg. A micon partition. In step 5, dialyzed fractions 5 and 6 from a 17-hr, 30% sucrose cushion were subjected to partition in an Amicon concentrator by sequential filtration through PMIO and XM300 membranes (Amicon). Aliquots were recovered after each filtration step and assayed for HBeAg. Biogel A5-M chromatography. In step 6, instead of a Sephadex G-200 column, a Bioge! A5M column was used according to the method previously described. Aliquots of the dialyzed 20%40% ammonium sulfate pellet were applied with PBSt as the running buffer. Fractions of 2.0 ml were collected, concentrated with Lyphogel, and assayed for HBeAg. DEAE-Sephadex chromatography. In step 7, instead of either Sephadex G-200 or Biogel A5M columns, a DEAE-Sephadex column was prepared. The same starting material as that described in step 6 was used, dialyzed extensively against the appropriate buffer, and applied to a column (60-ml bed volume) of Whatman DE52 (Whatman Inc., Clifton, N.].) with dimensions of 2.7 em X 17 em. In the first trial, phosphate was used as the anion on the column with 0.01 M sodium phosphate, pH 7.4, as the eluent. Stepwise elution from 0.1 M to 0.5 M phosphate by 0.1 M increments, followed by additional steps at 1.0 M and 3.0 M phosphate, was carried out. A second trial employed 0.01 M Tris-H'Cl buffer, pH 7.4, with chloride as the column anion. Gradient elution from 0.1 M to 0.5 M NaCI was followed by stepwise elution at 1.0 M and 3.0 M N aCl. At least 50 mg of protein (as determined by the method of Lowry et al. [13]) was applied to the column in each trial. Chromatography on a DEAE-Sephadex column was tried again in step 8 after an intervening separation on Sephadex G-200. Both elution techniques were used as described above.
Wei! et al.
Partial Characterization of HBeAg
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E
200,000 daltons, as shown both by its dispersion in a 20%-50% sucrose gradient and by its elution pattern during Biogel A5-M column chromatography. As Biogel A5-M is generally capable of resolving proteins
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ium sulfate precipitation was next passed over the column to fill all available nonspecific binding sites even after inactivation with ethanolamine. Approximately 1.5 mg bound. When an HBeAg preparation containing 9.6 mg of protein and 5.85 mg of IgG was passed over the column, all the HBeAg as well as >99% of the total protein and IgG was recovered. Thus, the HBeAg preparation contained no detectable activity against human immunoglobulins. An affinity chromatography column was prepared with anti-HBe as the ligand by coupling 116 mg of anti-HBe-IgG to a lO-ml volume of Sepharose 4B. Protein (31.2 mg) from a 20%-40% ammonium sulfate precipitate of normal human serum was then added to fill all of the available nonspecific binding sites. A sample (20 mg) of the same preparation of normal human serum was separately added to a 2.0-ml aliquot of the Sepharoseanti-HBe conjugate. Of the initial 20 mg, 99.9% was recovered from the pass-through volume and wash, a result indicating that no such nonspecific sites remained available. An HBeAg preparation was added to the remaining coupled Sepharose. As seen in table 2, the Sepharose-anti-HBe conjugate bound an equivalent amount of IgG and total protein as well as all of the detectable HBeAg in the preparation. Affinity chromatography with use of normal human serum containing rheumatoid factor as the ligand produced results similar to those obtained with GAHlgG. The column was preloaded with normal human serum to fill nonspecific binding sites. The rheumatoid factor column bound equivalent amounts of IgG and total protein along with all the HBeAg (data not shown). Several systems were employed in an attempt to decouple the HBeAg from the affinity chromatography columns for further analysis (table 3). All attempts were unsuccessful. As shown, reagents with pH values as low as 2.0 and as high as 10.9 were employed. All were allowed contact for 5-60 min with the respective columns. Separate studies were done to determine how long HBeAg would remain immunologically active in each decouplant. The times selected for each zompound were shorter than that which completely destroyed clearly discernible immunologic activity. After contact, each eluent was immediately titrated to pH 7.4 and dialyzed against
407
Partial Characterization of HBeAg
matography columns in sufficient quantities pre· vented both the further analysis of the IgG moiety and the use of affinity chromatography for further purification. The heterogeneity of charge, variability of weight, and behavior on affinity chromatography columns suggest that HBeAg is not simply IgG with an additional immunoreactive site, but that HBeAg and IgG are associated in a complex. These observations are in accord with the recent report of Takahashi et al. [II], who provided evidence that HBeAg can circulate in serum complexed to IgG or in free form. References 1. Magnius, L. 0., Espmark, J. A. New specificities in Australia antigen positive sera distinct from the LeBouvier determinants. J. Immunol. 109:1017-1021, 1972. 2. Perrillo, R. P., Gelb, L. D., Milligan, W. H. III, Wellinghoff, W., Aach, R. D. Discordant e antigen. DNA polymerase activity, and Dane particle responses in two patients representing an index case-contact case pair with hepatitis B virus infection. J. Infect. Dis. 136:117-121,1977. 3. Gitnick, G. L. [moderator], Goldberg, L. S., Koretz, R., Walsh, J. H. [discussants]. The liver and the antigens of hepatitis B. Ann. Intern. Med. 85:488-496, 1976. 4. Rebhun, R. J., Overby, L. R., Gitnick, G. L. Infectivity of asymptomatic e antigen positive and negative HBsAg carriers. Gastroenterology 71:925, 1976. 5. 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. 6. Neurath, A. R., Strick, N. Evidence against the postulated identity of e-antigen with DNA polymerase associated with the hepatitis B candidate virus. Intervirology 7:356-359, 1976. 7. Takahashi, K., Yamashita, S., Imai, M., Miyakawa, Y., Mayumi, M. Failure of antibody to e antigen to precipitate Dane particles containing DNA polymerase activity and hepatitis B core antigen. J. Gen. Virol. 38:431-436, 1978. 8. Gerin, J. L., Shih, J. W.-K., McAuliffe, V. J., Purcell, R. H. Antigens of hepatitis B virus: failure to detect HBeAg on the surfaces of HBsAg forms. J. Gen. Virol. 38:561-566, 1978. 9. Neurath, A. R., Strick, N. Host specificity of a serum marker for hepatitis B: evidence that "e antigen" has the properties of an immunoglobulin. Proc. Natl. Acad. Sci. U.S.A. 74:1702-1706,1977. 10. Vyas, G. N., Peterson, D. L., Townsend, R. M., Damle, S. R., Magnius, L. O. Hepatitis B "e" antigen: an
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weighing between 2 X 105 and 106 daltons [18], dispersion would result if a heterogeneous family of immunologically reactive molecules was involved. Rechromatography of the HBeAg peak on Sephadex G-200 demonstrated that it coelutes with the IgG immunoreactive material, which was but a small fraction of the total IgG present in the HBeAg preparation at that stage of the purification. Similarly, a GAHIgG-coupled affinity chromatography column bound both HBeAg and IgG, but not IgM. When rheumatoid factor was used as a ligand on an affinity chromatography column, 19G and HBeAg were bound. It should be noted that the preparations of HBeAg and antiHBe were themselves negative for rheumatoid factor. HBeAg appears not to be a monomeric IgG molecule by itself, since the immunoreactive HBeAg is larger than the IgG peak by column chromatography (figure 2). Likewise, HBeAg does not appear to be a multimeric IgG molecule since such molecules would appear in distinct peaks during Biogel A5-M chromatography. The data obtained by affinity chromatography suggest that there is a specific association between the two immunoreactivities (IgG and HBeAg), rather than that HBeAg has sufficiently similar biophysical and biochemical properties to copurify with normal human IgG. The heterogeneous nature of this association delineated by our studies corroborates the work of Neurath and Strick [9], who have reported that the HBeAg immunoreactivity of their preparations was spread over a pH range of 4.9-7.5 during isoelectric focusing. This observation implies a group of relatively negatively charged molecules and could explain our inability to elute HBeAg from DEAE-Sephadex despite the addition of up to a 3 M concentration of anion. The IgG associated with HBeAg could represent a specific class or subclass of IgG molecules, but no data are available to support.rhis hypothesis. IgG was probably retarded by the anti-HBe-coupled affinity chromatography column because it is presznt in a complex bound to HBeAg. HBeAg and IgG moieties lacking immunoreactivity could be removed from the affinity chromatography colLimns, as measured by protein content, only by highly chaotropic agents. The inability to de· couple the bound HBeAg from the affinity chro-
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12.
13.
15.
16.
17. 18.
of peptides and proteins to polysaccharides by means of cyanogen halides. Nature 214: 1302-1304,1967. Neurath, A. R., Trepo, C., Chen, M., Prince, A. M. Identification of additional antigenic sites on Dane particles and the tubular forms of hepatitis B surface antigen. J. Gen. ViroI. 30:277-285, 1976. Takahashi, K., Fukuda, M., Baba, K., Imai, M., Miyakawa, Y., Mayumi, M. Determination of e antigen and antibody to e by means of passive hemagglutination method. J. ImmunoI. 119:1556-1559, 1977. Pharmacia Fine Chemicals Catalogue, 1974. Pharmacia, Piscataway, N.J., 1974, p. 6-7. Tipton, C. L., Paulis, J. W., Pierson, M. D. Gel filtration of lipid mixtures. J. Chromatogr. 14:486-489, 1964.
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14.
apparent association with lactate dehydrogenase isozyme-5. Science 198:1068-1070, 1977. Takahashi, K., Imai, M., Miyakawa, Y., Iwakiri, S., Mayumi, M. Duality of hepatitis B e antigen in serum of persons infected with hepatitis B virus: evidence for the nonidentity of e antigen with immunoglobulins. Proc. NatI. Acad. Sci. U.S.A. 75:1952-1956, 1978. Kaplan, P. M., Greenman, R. L., Gerin, J. L., Purcell, R. H., Robinson, W. S. DNA polymerase associated with human hepatitis B antigen. J. ViroI. 12:9951005, 1973. Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. Protein measurement with the Folin phenol reagent. J. BioI. Chern. 193:265-275, 1951. Axen, R., Porath, J., Ernback, S. Chemical coupling-
