Download Antigens of Hepatitis B Virus: Failure to Detect HBeAg on the

J. gen. Virol.
56I
(I978), 38, 561-566
Printed in Great Britain
Antigens o f Hepatitis B Virus: Failure to Detect H B e A g on
the Surfaces of H B s A g Forms
( A c c e p t e d I2 S e p t e m b e r I977)
SUMMARY
The particulate forms of HBsAg were analysed for the presence of HBeAG on
their surfaces. By immunodiffusion analysis, anti-HBe did not form precipitin
bands with the purified forms of HBsAg and hyperimmune guinea pig antisera to
these forms did not react with HBeAg. Lines of non-identity were observed
between the HBeAg determinants (el and e~) and the Dane particles and filaments
isolated from an HBeAg-positive serum. Finally, anti-HBe failed to precipitate the
polymerase-positive subpopulation of Dane particles, indicating that anti-HBe has
no direct role in virus neutralization.
The virion of Type B hepatitis appears to be a 43 nm spherical particle (Dane particle;
Dane, Cameron & Briggs, I97O) with a complex morphology consisting of an outer lipoprotein coat and an internal 27 nm core. Two antigen systems appear to be specified by the
genome of hepatitis B virus (HBV) and are associated with structural components of the
Dane particle. The determinants of hepatitis B surface antigen (HBsAg) are expressed on the
lipoprotein coat of the Dane particle and other subviral forms found in the sera of chronically-infected individuals. The hepatitis B core antigen (HBcAg) is distinct from HBsAg and
is associated with the particulate a7 nm cores of the Dane particle (Almeida, Rubenstein &
Stott, I970, some of which contain an endogenous DNA polymerase activity (Kaplan et al.
I973, I976) and circular double-stranded DNA (Robinson, Clayton & Greenman, I974).
The hepatitis B e-antigen (HBeAg) and its antibody (anti-HBe) represent yet another
antigen system in association with Type B hepatitis and not other liver diseases (Magnius &
Espmark, I972) including Type A hepatitis (Maynard et al. I976). The HBeAg complex
appears to consist of more than one specificity (Williams & LeBouvier, I976) and physical
characterization (Magnius, I975) indicated that it is composed of soluble proteins of approx.
3ooooo mol. wt. A number of studies demonstrated that the presence of HBeAg correlates
well with Dane particles (Nielsen, Dietrichson & Juhl, I974) and HBV-specific DNA
polymerase activity (Nordenfelt & Sandberg, I976; Takahashi et al. I976) in serum and that
these markers are important indicators of HBV infectivity (Alter et al. I976; Okada et al.
x976). Despite the specific association of HBeAg with Type B hepatitis, most authors have
cautioned that the evidence is not yet conclusive that it respresents a HBV gene product;
HBeAg might also represent a specific response of the host to HBV replication (El Sheikh et
al. I975; Magnius et al. I975). However, in a recent report, Neurath et al. 0976) concluded
that HBeAg was exposed on the surface of Dane particles and tubular forms of HBsAg and
was not present on the surface of the approx. 2o nm form. This demonstration of HBeAg as
a protein associated with the structure of the presumptive hepatitis B virus supports the
possibility that it represents a virus gene product and has significant implications for
approaches to prophylaxis. Thus, it predicts that anti-HBe might have a direct or additive
role in virus neutralization and might provide a basis for the use of anti-HBe in passive
immunization as suggested by Okada et al. 0976). Current vaccine programs (Purcell &
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Gerin, I975; Buynak et al. I976), which are based on the use of the 2o nm subviral form of
HBsAg, would have to be evaluated on the basis that the immunogen lacks a potentially
important surface antigen of the HBV which is involved in virus neutralization. Because of
these important implications, we attempted to confirm the findings of Neurath et al. (I976)
using other methods of analysis but failed to find evidence for the presence of HBeAg on the
Dane particle surface.
Two types of analysis were used in this evaluation: immunodiffusion in agarose gels and
double-antibody immunoprecipitation of Dane particles as measured by the endogenous
DNA polymerase activity. The plasma of a chronic HBsAg/ayw carrier (NCB), positive for
HBsAg-specific DNA polymerase activity and HBeAg, was used as a source of 'heavy'
Dane particles (Kaplan et al. 1976). The Dane particles were isolated by centrifuging the
plasma for 4 h at zoooo rev/min in the Spinco zI rotor, resuspending the pellet in phosphate
buffered saline (PBS; o'85 % NaC1, o.oI M-phosphate, pH 7"4) to I/2o volume, followed by
re-centrifuging and resuspending the pellet in I/5o of the original volume. Electron microscopic examination of the preparation, designated NCB P-z, revealed Dane particles and
filaments as the predominant particulate forms. HBsAg was also purified from the plasma of
a chronic HBsAg/ayw carrier (Ytr) by zonal centrifugation procedures as previously
described (Gerin, Holland & Purcell, i97i ; Gerin, I973). The rate sedimentation profile (see
Gerin, i973, Fig. I5) of twice CsCl-banded HBsAg revealed the usual 2o nm forms as well as
filamentous forms which sedimented further in the sucrose gradient. Three pools were made
from the gradient fractions: pool one contained 2o nm forms of HBsAg; pool two, the long
filamentous forms plus the low-density (I.2o g/ml) Dane particles (Gerin, Ford & Purcell,
I975); and pool three, short filamentous forms found in gradient fractions intermediate
between those which made up pools one and two. Each pool was re-banded in CsC1 and
HBsAg recovered at z.2o g/ml. After dialysis against o-o1 ~a-tris-HC1 buffer, pH 7"4, the
individual pools were used to hyperimmunize guinea pigs as previously described (Purcell et
al. I97o). Each guinea pig was inoculated in the hind footpad at time zero with a dose of
2o/zg of HBsAg, I : I in Freund's complete adjuvant. At 4 and 6 weeks, each animal was
again inoculated intraperitoneally with 2o/zg of aqueous HBsAg and exsanguinated by heart
puncture Io days after the 6-week boost. The sera of individual guinea pigs were assayed for
anti-HBs by passive haemagglutination (PHA; Vyas & Schulman, I97o) using commercial
reagents (Electronucleonics, Bethesda, Maryland). When tested against red blood cells
coated with an ayw antigen, the hyperimmune sera to pools one, two and three titred
I:320OOOO, I:64OOO0 and I:512ooo , respectively. The standard HBeAg reagent was
obtained by plasmapheresis of a chronic HBsAg/adw carrier chimpanzee and contained
both the el and e2 specificities (Williams & LeBouvier, I976). The standard anti-HBe was
obtained from a human chronic carrier, specified both anti-HBel and HBe2, and also
contained anti-HBc activity. The anti-HBc reagent (795) was from a chronic HBsAg carrier
and has been previously characterized (Moritsugu et al. I975; Budkowska, Shih & Gerin,
I977); it contained HBeAg and therefore lacked anti-HBe. The human anti-HBs serum was
obtained from a donor convalescent from Type B hepatitis and was predominantly antiHBs/a.
Immunodiffusion analyses were performed by the procedure of Williams & LeBouvier
0976) with o'4% agarose (L'Industrie Biologique Fran~aise, Gennevilliers, France) in
o'I5 M-NaC1 and tris-HC1, pH 7.6. Wells, 3 mm in diam., were cut in the agarose and filled
with approx. 2o #1 of sample; wells loaded with the standard HBeAg and anti-HBe reagents
were refilled twice within the first few hours. All other wells were filled once. Guinea pig
antisera to the HBsAg forms were absorbed with normal human serum before analysis. The
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1
3
Fig. t. Non-identity is demonstrated between the HEeAg antigenic determinants (e, closer to the
antibody well, e~ closer to the antigen well) and the Dane particle-anti-Dane precipitin band. The line
of precipitation between wells 3 and 4 represents an HBsAg-anti-HBsAg reaction. In test patterns
(not presented here) in which the positioning of wells was changed, this line shows partial identity
with the Dane-anti-Dane precipitin band. The spurring reaction in this case results from the subtype
difference between the I-r£BsAgin the HBeAg positive serum (subtype adw) and the Dane particle
preparation (subtype ayw). Well i, Dane particle preparation (subtype ayw); well 2, anti-Hl3eAg
(anti-el and anti-e2) serum; well 3, guinea pig anti-Dane and long filament serum; well 4, Hl~eAg
(el and e~), HBsAg subtype adw) positive serum.
Fig. 2. Electron micrograph of the precipitin band formed by the reaction of anti-Hl3s with the
NCB P-2 preparation. The precipitin band was excised from the agarose gel, extracted with PBS
and examined in an Hitachi H U I I E after negative staining with 1% phosphotungstic acid.
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Table I. Failure of antibody to HBeAg to precipitate DNA polymerase-positive Dane
particles
DNA polymerase activity
remaining in supernatant*
Antiserum
Human
Normal serum
Anti-IrA3s
Anti-HPc
Anti-HPe
Guinea pig
Preserum
Anti-pool one (2o nm)
Anti-pool two (Dane particles
& filaments)
Anti-pool three (short
filaments)
~H, ct/min
P/N~
I59I
138
i 742
1567
I r9
i .o
13"o
I 1.7
1752
82
17"7
0'8
9I
o'9
9o
o'9
* Dane particle concentrate (NCB P-2; 25 #I) was incubated with 20 #1 of I : 5 dilution of antiserum for
18 h at 4 °C at which time 75/zl of an optimum concentration of second antibody (goat anti-human IgG or
rabbit anti-guinea pig IgG) was added. After 2"5 h at room temperature the mixture was centrifuged at 3ooo g
for 30 min. The supernatant (75 #1) was solubilized by the addition of IO #1 of 1% Nonidet P-4o and 1%
fl-mercaptoethanol. Polymerase reaction mixture (4o #1) was added to yield a final concentration of
o.I28 M-tris-HCl, pH 7'5; MgCI~, 32 raM; NH4C1, 96 rr~; o'4 mM each of dATP, dCTP and dGTP, and
1'25 #Ci of 3H-TTP, sp. act. 45Ci/mmol (New England Nuclear). A portion (4o #1) of the mixture was
withdrawn at zero time and after 3 h at 37 °C and processed for counting as described by Kaplan et al. (I973).
The zero time values ranged from 3o to 4o ct/min.
t P/N means positive/negative and represents the 3H ct/min value of the experimental sample divided by
that obtained using either normal human serum (134 ct/min) or guinea pig serum (99 ct/min) in place of the
NCB P-2 concentrate.
plates were stored in a humid chamber at r o o m temperature and examined daily for up to 4
days. Precipitin lines were observed between the three H B s A g pools and the respective
guinea pig antisera. Each individual antiserum showed a line o f identity with all three
H B s A g pools and a reference HBsAg/ayw serum, and partial identity with the HBsAg/adw
in the standard H B e A g serum. The reaction between the standard H B e A g serum and the
anti-HBe reagent revealed lines (el and e~) o f non-identity with the HBsAg-anti-HBs line; no
anti-HBel or anti-HBe2 activity was detected in any of the guinea pig antisera to the various
H B s A g forms. Likewise, the anti-HBe reagent failed to react with the three pools of purified
HBsAg. Finally, reactions o f complete non-identity were observed between the H B e A g (el
and e~) determinants and the H B s A g precipitin line formed from the reaction o f the D a n e
particle (NCB P-2) preparation with the guinea pig antiserum to pool two (Dane particles
and filaments; Fig. I). In order to be certain that the H B s A g precipitin line was not due to
the presence o f residual 2o n m forms, the precipitin line was excised from the gel, extracted
in PBS buffer with mechanical disruption, and examined by electron microscopy after
negative-staining with 1% phosphotungstic acid. The morphological forms (Fig. 2) participating in the H B s A g reaction consisted of D a n e particles and filaments. The reaction o f
non-identity between these particles and the el and e2 specificities o f H B e A g clearly indicated
that these specificities were not present on the surfaces o f these particulate forms.
The N C B P-2 preparation contained endogenous D N A polymerase activity which was
precipitated by anti-HBs and not by anti-HBc or normal serum (Table I). These criteria
establish the activity as a marker o f the subpopulation o f intact Dane particles considered to
be the infectious hepatitis B virus. The standard anti-HBe reagent failed to precipitate the
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565
polymerase activity indicating that the HBelAg and HBe2Ag specificities were not exposed
on the surface of the presumed HBV. The guinea pig antisera to the various HBsAg forms
precipitated the polymerase activity as expected from their high anti-HBs titres.
In summary, the conclusion that the determinants of HBeAg are not expressed on the
surfaces of the HBsAg particles is based on the following lines of evidence: anti-HBe did not
react with any of the purified particulate forms and hyperimmune guinea pig antisera to
these forms failed to react with the reference HBeAg; clear lines of non-identity were
observed between the HBeAg determinants and the Dane particles and filaments isolated
from an HBeAg-positive serum; and the failure of anti-HBe to precipitate the polymerasepositive Dane particles using a sensitive double antibody radioimmunoprecipitation assay.
Whether the HBeAg complex is coded for by the HBV genome or represents a specific
response of the host to HBV infection still remains an important question. Lam, Tong &
Rakela (~977) reported that the treatment of Dane particle-rich preparations with o'5 %
Tween 8o released HBeAg suggesting that HBeAg represents an internal structural protein
of the HBV. On the other hand, Neurath & Strick (2977) have found that HBeAg has
properties of an immunoglobulin and suggested it represents an idiotypic antibody; clearly a
host response to HBV infection. The earlier results (Neurath et al. ~976) were explained by
the possibility that a rheumatoid factor might have been present in their anti-HBe reagent.
However, another report from the same laboratory (Trepo et al. 2976) localized HBeAg to
the cytoplasm of infected hepatocytes. In any case, we report here that the determinants of
HBeAg are not expressed on the surfaces of Dane particles, filaments or 2o nm HBsAg
forms. Significantly, HBeAg was not found on the surface of the population of Dane particles
that contains DNA polymerase and correlates with HBV infectivity indicating that anti-HBe
has no direct role in virus neutralization. Therefore, the correlation between anti-HBe and
lack of infectivity is probably an indirect one; if so, there appears to be no basis for active
immunization for anti-HBe or the passive administration of anti-HBe as prophylactic
measures for Type B hepatitis.
The Rockville Laboratory of the Molecular Anatomy Program is supported by the NIAID
under Union Carbide's contract with the U.S. Energy Research and Development Administration. We are indebted to Mr Terry Popkin for producing the photograph of the
immunodiffusion plate.
Molecular Anatomy Program
Oak Ridge National Laboratory
564o Fishers Lane
Rockville, Maryland 2o852
JOHN L. GERIN
J. WM-Kuo SHIn
Laboratory of Infectious Diseases
National Institute of Allergy and Infectious Diseases
National Institutes of Health
Bethesda, Maryland 2ooi4, U.S.A.
VINCENT J. McAuLIFFE
ROBERT H. PURCELL
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