Immunochemical structure of the carboxy-terminal part of ... - CiteSeerX

Journal of General Virology (1993), 74, 1335 1340. Printedin Great Britain

1335

Immunochemical structure of the carboxy-terminal part of hepatitis B e antigen: identification of internal and surface-exposed sequences M a t t i S~illberg, 1,2. Peter Pushko, 3 Ivar Berzinsh, 3 Vadim Bichko, 3 Peter Sillekens, 4 M i c h a e l N o a h , s Pauls Pumpens, 3 Elmars Grens, 3 Britta Wahren ~ and Lars O. M a g n i u s 2 2Department of Virology, The National Bacteriological Laboratory, S-105 21 Stockholm, 1 Department of Clinical Virology, Huddinge University Hospital, F69, S-141 86 Huddinge, Sweden, a Institute of Molecular Biology, Latvian Academy of Sciences and University of Latvia, Krustpilst Str. 53, Riga, Latvia, 40rganon Teknika, 5280 AB Boxtel, The Netherlands and ~Behringwerke GmbH, Marburg, Germany

The C-terminal region of hepatitis B virus (HBV) e antigen (HBeAg), amino acids (aa) 12I to 147, was characterized for reactivity with 15 monoclonal antibodies (MAbs) and sera from 16 chronic carders on the HB surface antigen with anti-HBe. Recombinant proteins exposing fragments of the HBc/e polypeptide (aa 29 to 176, 60 to 176, 101 to 176, 121 to 176, 134 to 176, 138 to 176, 139 to 176, 140 to 176, 146 to 176 and 156 to 176) fused to the N terminus of the coat protein of RNA phage fr were constructed, as were two sets of synthetic peptides covering residues 121 to 136 and 130 to 147, where each residue was sequentially substituted by alanine. The MAbs were found to recognize overlapping epitopes in the fusion proteins within residues 121 to 176; however, none of the MAbs reacted with proteins covering residues 146 to 176 and 156 to 176.

Using the synthetic peptides it was found that the MAbs recognized epitopes at residues 128-TPPAYR- 133, 133RPPNAP-138, 135-PNAPIL-140, 138-PILSTLPE-145 and 143-LPET-146. Only MAbs recognizing the epitope 128-TPPAYR-133 were found to react with both native HBeAg and denatured HBcAG, whereas MAbs recognizing epitopes located closer to the C terminus of HBeAg were reactive only with denatured HBcAg. The recognition sites for the human IgG1 overlapped with the epitopes of the MAbs recognizing native HBeAg. Our interpretation of these findings is that the region 124 to 133 is on the surface of native HBeAg and denatured HBcAg, and that the adjacent region 135 to 147 is not accessible on the surface of native HBeAg, but becomes exposed on denatured HBcAg.

Introduction

contains a signal sequence that guides the protein to the endoplasmic reticulum, where the precursor HBeAg residues - 2 9 to - 1 1 are cleaved off (Standing et al., 1988). After another proteolytic cleavage, between aa 149 and 150, the remaining part is secreted into the circulation (Ou et al., 1986). Thus, serum HBeAg is considered to consist of aa - 1 0 to - 1 of the precore region (Bruss & Gerlich, 1988) plus residues 1 to 149 of HBcAg (Takahashi et al., 1983). However, HBeAg containing the whole precore region has also been found in human sera (Takahashi et al., 1992). Two major antigenic regions of HBeAg have been identified (Imai et al., 1982; Ferns & Tedder, 1984; Salfeld et al., 1989). Salfeld et al. (1989) described an antigenic region within the HBc/eAg sequence which resided around aa 130 to 138 but was suggested to require the presence of a part of the N-terminal region of the HBcAg sequence in order to be recognized as a fusion protein (HBe2; Salfeld et al., 1989). However, it has been shown that monoclonal antibodies (MAbs) and human sera may recognize the region around aa 130 as a linear

Hepatitis B e antigen (HBeAg), originally described by Magnius & Espmark (1972), is a secretory protein that occurs in soluble form in sera from individuals infected with hepatitis B virus (HBV) and signals a contagious state of the disease (Alter et al., 1976; Okada et at., 1976; Bonino et al., 1991). The function of HBeAg has been suggested to be the protection of HBV-infected hepatocytes from the host immune surveillance (Uy et al., 1986; Milich et al., 1990; Bonino et al., 1991). HBeAg shares 149 amino acids (aa) with the HBV nucleocapsid protein, immunologically defined as the core antigen (HbcAg). HBeAg determinants can be revealed on native HBcAg by treatment with detergent or by limited proteolysis (Budkowska et al., 1979). The first A U G in the C-terminal open reading frame corresponds to the start of transcription of the mRNA for the HBeAg precursor while the second in-frame A U G corresponds to that of HBcAg. Intracellular HBeAg including the precore residues - 2 9 to - 1 0001-1494 © 1993SGM

1336

M . Siillberg and others

epitope which has been mimicked by synthetic peptides ( S / i l l b e r g et al., 1 9 9 1 a , c; B i c h k o et al., 1992). I n t h i s paper, we report on the immunochemical characterization of the C-terminal part of the HBeAg at the resolution of a single amino acid.

Methods Human HBeAg, anti-HBe and anti-HBc positive sera. A total of 22 human sera from chronic carriers of HBsAg were selected from consecutive sera sent to the Department of Virology at the National Bacteriological Laboratory, Stockholm from 1988 to 1992. All sera were serologically defined as regards HBV status using commercial radioimmunoassays (RIAs; Abbott). Six of the sera were found to be positive for HBsAg, HBeAg and anti-HBc in these RIAs, and 16 were found to be positive for HBsAg, anti-HBe and anti-HBc. All of the 16 sera that were positive for anti-HBe in RIA were also found to be reactive to a peptide covering residues 121 to 140 of HBeAg (S~illberg et aL, 1991a). MAbs. The production and characterization of the MAb to HBec~ (141/03) and HBefl-specific MAbs (57/8, 141/158 and 141/207) have been described elsewhere (Noah & Harthus, 1987; S/illberg et al., 1991 a, b). The MAbs were obtained by screening primary hybridomas for activity in the Enzygnost anti-HBe assay (Behringwerke). The MAbs HBeOT6P and HBeOT7Q were raised against recombinant HBcAg (rHBcAg, subtype adw; Ono et at., 1983). The primary hybridoma clones were screened for MAb production by enzyme immunoassays (EIAs) using recombinant HBeAg (rHBeAg; Biogen) or rttBcAg (Chiron Corporation, P. Sillekens et al., unpublished). They will be referred to as MAbs against rHBeAg in the following text. The MAbs 14Ell, 14G3, 14E12, 13D9, 13B1, 14D5, 10F10, 13C9, 10C6 and 13D3 were raised against SDS-denaturated rHBcAg (drHBcAg of ayw subtype; Bichko et al., 1985, 1992), which also was used for the screening of primary hybridomas. These will be referred to as MAbs against drHBcAg in the following text. Characterization of MAbs in commercial assays. The MAbs were tested for anti-HBc and anti-HBe specificity using Corab and HBe RIA (Abbott). All HBe MAbs were also analysed for HBe specificity using the semi-automated Abbott HBe IMX system. In the anti-HBc and anti-HBe RIAs, and in the anti-HBe IMX assay, all MAbs were diluted 1 : 100 in the negative control serum provided with the assay, and the mixture was then tested for its ability to inhibit binding to 125I-labelled human anti-HBc and anti-HBe according to the manufacturer's instructions. Reactions giving > 50% inhibition of radiolabelled antibody are regarded as positive. In the anti-HBe IMX assay, a sample to cut-off ratio (S/CO) less than 1.0 is regarded as reactive. HBcAg-fr coat protein (CP) fusion proteins. The amino acid numbering is given according to translation starting at the second AUG. A set of fusion proteins containing HBcAg successively truncated from the N to C terminus was constructed by unidirectional shortening of the C gene fragment (encoding aa 29 to 176) cloned into the polylinker at a position between aa 1 and 2 of the RNA phage fr CP gene (frCP). The latter has been expressed in Escherichia coli under control of the tryptophan operon promoter (Kozlovskaya et al., 1986). Restriction endonuclease cleavage sites at the 5' part of the polylinker were used for unidirectional truncation of the HBeAg gene fragment by using nucleases Exo III and S1, according to the method of Henikoff (1984). Proteins containing in-frame deletions were expressed in E. coli from recombinant plasmids and selected by Western blotting with rabbit antibodies against frCP. The sequences of the recombinant genes

were analysed by the dideoxynucleotide chain termination method (Sanger et al., 1977). Proteins were separated by SDS-PAGE (Laemmli, 1970). Western blotting was performed essentially as described by Towbin et al. (1979). Blotted nitrocellulose membranes were incubated overnight at 4 °C with various dilutions of the MAbs. Bound MAbs were detected by incubation with horseradish peroxidase (HRPO)-conjugated rabbit anti-mouse Ig (Sigma) for 2 h at 37 °C followed by the addition of odianisidine substrate.

Solid-phase peptide synthesis. The peptides 121SFGVWlRTPPAYRPPN-136 and 130-PAYRPPNAPILSTLPETT147 (HBV subtype ayw; Galibert et al., 1979) and analogues thereof, in which each amino acid within residues 124 to 135 and 131 to 146 was sequentially substituted by alanine or glycine, were synthesized according to a previously described method (S/illberg et al., 1991 c). The peptides were dissolved in water at a concentration of 1 mg/ml. When analysed by HPLC, peptides were found to be at least 70 % pure and were used without further purification. Peptide-based EIAs. Peptides were coated onto microtitre plates at a concentration of 1 ~tg/ml in 0-5 M-sodium carbonate buffer pH 9.6 and kept overnight at 4 °C. The plates were incubated for 1 h at 37 °C with MAbs, diluted 1:1000 and 1:10000 in PBS with 0.05% Tween 20 (PBST), 0'5 % BSA and 10% fetal calf serum. Bound MAbs were then detected by HRPO-conjugated rabbit anti-mouse Ig (Dakopatts) diluted 1 : 3000 in PBST with 0.5 % BSA. After a 1 h incubation at 37 °C with the conjugate, bound conjugate was measured by incubation with o-phenylenediamine (Dako). Absorbances were read at 492 nm. All human sera were tested with peptide-coated plates, at a dilution of 1 : 100 in PBST with 1% BSA and 2 % goat serum. Bound serum IgG1 was located by a MAb to human IgG1 (clone NL16; Unipath) for 1 h at 37 °C. Bound MAb was then assayed as described above. Assays for MAbs" ability to bind to recombinant and native HBeAg. The binding of MAbs to denaturated recombinant HBcAg was performed according to a method previously described (S~illberg et al., 1989) in which SDS-treated HBcAg was coated onto microplates and assayed as described above. The binding of MAbs to native HBeAg was performed using soft microtitre plates (Dynatech) coated with the different MAbs. To each MAb, 50 gl portions of recombinant HBeAg (neutralizing reagent, HBe RIA, Abbott) and of each of the six human sera containing native HBeAg (diluted 1:100) were added. These were incubated with the MAbs for 90 min at 37 °C. The plate was washed and incubated with radiolabelled human anti-HBe (HBe RIA) to reveal bound HBeAg. The plates were then cut and the radioactivity in each well was measured in a ?,-counter. Sample to negative ratios were obtained by dividing the c.p.m, of each individual HBeAg sample with the c.p.m. of each well to which HBeAg-negative sera (HBe RIA) had been added. The cutoff was set at the same level as that for the commercial HBeAg RIA, 3.1 times the c.p.m, of the HBeAg-negative sera.

Results Characterization o f M A b s in a n t i - H B c a n d a n t i - H B e assays The ability of MAbs to inhibit binding of human antiHBc was assayed in Corab (Abbott), and none of the MAbs showed any inhibition of human anti-HBc. Inhibition of human anti-HBe was assayed using the competitive HBe RIA or HBe IMX (Abbott). Only the M A b s t o HBec~ ( 1 4 1 / 0 3 ) a n d HBe/~ ( 5 7 / 8 , 1 4 1 / 1 5 8 ,

HBeAg C-terminal structure

(a) ~

(c)

(b)

(~)

1337

(e)

30K R

20K

-

15K-

Fig. 1. Reactivityof the MAbs with the HBcAg-frCP recombinant proteins in Western blotting: (a) MAb 57/8 to HBefl(b) MAb HBeOT7Q to rHBeAg, (c) MAb 14Ell to drHBAg, (d) MAb 10C6 to drHBcAg and (e) MAb 13D3 to drHBcAg. 3

$26 (preS2)

/

2

Jtl

.................. ,.__,Ill, 14Ell (drHBc) I 14G3 (drHBc)

0

Epitope mapping using fusion proteins

HBeOT7Q (rHBeAg) t

[ HBeOT6P(rHBeAg)

J

,,I _.,_._,tll,11 I

14E12(drHBc)

]

illll,1,t llllilliill,,IIl,lnililIII,ti IIIII,.,lll,lllllll,.,II1,1111111. II1,1. !1111[ol [..lilill . Illll[,ll_.[lllllllllllll ...... ,I I --

I

1

13 (

Bc)

10F10(~h~l~Bc)

I

13B (dr~IBc)

I

14 (L-HBc)

]

13C~9(~tBc)

]

10~-6"(drl~13c) I

13D3 (drHBc)

~ AYRPPNAPtLSTLPET

-AYRPPNAPILSTLPE1 2 1 o

i

t

Ihllllllhh..J -AYRPPNAPI LSTLPET

Substituted residue (by A or G)

Fig. 2. Fine mapping of the MAbs using peptides, residues 130 to 147, in which residues were sequentially substituted by alanine or gtycine. The respectiveMAbs are indicatedin each panel. The MAb $26 specific for the HBV preS2 region served as a negative control. 141/207), showed significant inhibition of human antiHBe, as described in a previous report (Sfillberg et al., 1991 a).

The reactivity of MAbs in Western blotting was tested with the fusion proteins containing fragments 29 to 176, 60 to 176, 101 to 176, 121 to 176 and 156 to 176 of the HBcAg. All MAbs recognized the proteins truncated to region 121 to 176 of the HBcAg. None of the MAbs recognized fusion protein containing aa 156 to 176 (data not shown). Fusion proteins containing HBcAg fragments 101 to 176, 121 to 176, 134 to 176, 138 to 176, 139 to 176, 140 to 176, and 146 to 176 were then used for epitope mapping. The shortest protein reactive with the MAb 57/8 contained aa 121 to 176 (Fig. la). The MAb HBeOT7Q against rHBeAg and MAb 14Ell against drHBcAg recognized proteins covering residues 101 to 176, 121 to 176 and 134 to 176 (Fig. lb, c). The MAb 10C6 against drHBcAg recognized proteins containing fragments 121 to 176, 134 to 176 and 138 to 176 (Fig. 1 d), and the MAb 13D3 against drHBcAg recognized fragments 139 to 176 and 140 to 176 (Fig. 1 e). Additional bands at low M r are likely to be intracellularly degraded HBc-containing products.

Fine mapping of MAb epitopes by synthetic peptides Fine mapping of the MAbs to HBefl (57/8, 141/158 and 141/207) has previously been performed by overlapping peptide and substitution analogue sequences (S/illberg et al., 1991b). The epitopes were shown to consist of aa 128-TPPAYR-133 (57/8 and 141/207) and 129-PPAY132 (141/158).

M. Siillberg and others

1338

T a b l e 1. Ability of MAbs to bind to undenatured recombinant and native human (A to F) HBeAg in a microtitre-based HBeAg capture RIA Binding (c.p.m. in RIA)*

MAb coated on solid phase 141/03 141/158 14El1 14G3 10C6 1303

Specificity or raised against NCt HBe7 HBefl drHBcAg§ drHBcAg drHBcAg drHBcAg

20 20 10 8 16 7

HBeAg-positive human serum rHBeAg~

A

B

C

D

E

F

726 (36.3) 1658 (82.9) 7 (0.7) 4 (0.5) 8 (0.5) 3 (0.4)

947 (47.4) 1225(61.2) 28 (2.8) 7 (0.9) 18 (2.2) 3 (0.4)

103 (5.2) 191 (9-6) 4 (0.4) 15 (1-9) 15 (1.9) 3 (0.4)

836 (41-8) 856 (42-8) 36 (3"6) 22 (2.8) 25 (3.1) 14 (2.0)

312 (15.6) 549 (27.4) 8 (0.8) 9 (1.1) 10 (1.2) 8 (1.1)

68 (3.4) 156 (7.8) 19 (1.9) 7 (0.9) 10 (1.2) 11 (1.6)

501 (25.0) 467 (23.4) 15 (1.5) NTH 19 (2.4) NT

* The sample to negative ratios given in parentheses are calculated by dividing the sample c.p.m+ with the c.p.m, of the negative control used in each MAb assay. ~ NC, The negative control serum from HBeRIA (Abbott). rHBeAg supplied as the neutralizing reagent in HBe RIA (Abbott). § drHBcAg, Denatured recombinant HBcAg. II NT, Not tested.

2105

541

100

100

:tilt U lid,.+l,IL i

Source/ specifity of antibody

+oi'll'l ........... I.I.I ;IIIII,, +iIIIII ............. IIi ,, 0

.9

348

100

0

m..m..m

VWI RTPPAYRPPN

I

0

50

0

0

7350

~

-VWIRTPPAYRPPN

I00=

. 5o

0

VWl RTPPAYRPPN

509

m I00

...........

..l VWI RT PPAYRPPN

mnnn

Human •

-VWIRT P PAYRPPN 2202

5

+ -VWIRT PPAYRPPN

Substituted residue within residues 124 to 135 of HBc/eAg Fig. 3. Characterization of six of the 126 human sera with IgG 1 binding to HBe residues 121 to 136 using peptide analogues in which residues were sequentially substituted by alanine or glycine. Values are given as a percentage of residual binding to the substitution peptide analogue, as compared with the original peptide.

anti-ttBe/] rHBeAg drHBcAg

ld

Reactivity with native denat. HBeAg HBcAg

Amino acid residue of H B e A g 125

130

135

S FGV~VI RT PPAYRPPNAP

140

145

I L S TL P ETT + + + +

+

++

+ + + + + + + + + + + + + + -

+ + + + + + + + + + + + + + + + +

-

-

-/+

-/+

T h e p e p t i d e 1 3 0 - P A Y R P P N A P I L S T L P E T T - 1 4 7 , representing the C - t e r m i n a l p a r t o f H B e A g ( w i t h o u t the two C - t e r m i n a l valine residues), as well as its s u b s t i t u t i o n a n a l o g u e s were tested in E I A with the M A b s a g a i n s t r H B e A g a n d d r H B c A g recognizing this region, acc o r d i n g to e p i t o p e m a p p i n g with the fusion p r o t e i n s d e s c r i b e d above. The M A b s r e c o g n i z e d o v e r l a p p i n g e p i t o p e s 1 3 3 - R P P N A P - 1 3 8 , 1 3 5 - P N A P I L - 1 4 0 , 138-PILS T L P E - 1 4 5 a n d 143-LPET-146 (Fig. 2).

Binding of MAbs to native HBeAg T h e ability o f M A b s c o a t e d o n t o m i c r o p l a t e s to b i n d h u m a n H B e A g is s h o w n in T a b l e 1. A s shown, o n l y M A b s to H B e e (141/03) a n d to HBefl (141/158) are clearly c a p a b l e o f b i n d i n g b o t h native a n d r e c o m b i n a n t

+ + +

+

+ + + + + + + +

Fig. 4. Summary of human IgG1 and MAb recognition of HBeAg residues 121 to 147. Each filled square indicates a residue that cannot be changed to alanine or glycine and retain antibody binding. The reactivity of each serum IgG1/MAb with native HBeAg and denatured HBcAg is shown on the right.

H B e A g , when c o a t e d o n t o m i c r o p l a t e s . N o n e o f the M A b s raised a g a i n s t r H B e A g ( d a t a n o t shown) o r d r H B c A g ( 1 4 E l l , 14G3, 10C6 a n d 13D3), show m o r e t h a n b o r d e r l i n e reactivities w i t h s o m e o f the H B e A g c o n t a i n i n g sera (Table 1). N o n e o f the o t h e r M A b s recognizing o v e r l a p p i n g e p i t o p e s w i t h i n residues 130 to 147 b o u n d to s e r u m - d e r i v e d H B e A g ( d a t a n o t shown).

H B e A g C-terminal structure Mapping o f recognition sites o f the C-terminal H B e region

The IgG1 reactions for six of the 16 human sera, with the substitution peptide analogues of region 121 to 136, are given in Fig. 3. A summary of the IgG1 recognition sites of the 16 human anti-HBe positive sera is given in Fig. 4, in relation to the epitope locations of 14 MAbs to the Cterminal domain of HBeAg. All sera recognized various sites within the region 124 to 133, whereas only MAbs to HBefl (57/8, 141/158 and 141/207) show recognition sites corresponding to the ones of the human sera. A summary of the ability of each serum IgG1 and MAb to bind native HBeAg and denatured HBcAg is also given in Fig. 4. As shown, human antibodies and MAbs with similar recognition sites bind serum HBeAg, whereas the MAbs against rHBeAg and drHBcAg do not. However, all sera and all MAbs show strong reactivity to drHBcAg (data not shown).

Discussion Two major HBe determinants have been observed by several authors (Imai et al., 1982; Ferns & Tedder, 1984; Salfeld et al., 1989). Mutual correspondence of these determinants termed HBe-a and HBe-b (Imai et al., 1982), e-e and e-fl (Ferns & Tedder, 1984) or HBel and HBe2 (Salfeld et al., 1989) has not been investigated thoroughly. It may be assumed that HBe-a, HBe~ and HBel constitute one group of similar or identical determinants residing around aa 80 of the HBeAg whereas HBe-b, HBefl and HBe2 form the other group at its carboxy-terminal part (Salfeld et al., 1989; Sfillberg et al., 1991a, b; Bichko et al., 1992). Here we report the immunochemical identification of several overlapping but functionally distinct epitopes at the carboxy-terminal part (residues 124 to 146) of the HBeAg using recombinant proteins and synthetic peptides. Based on the findings reported herein, we consider the MAbs against rHBeAg (HBeOT6P and HBeOT7Q) and drHBcAg (14Et 1, 14G3, 14E12, 13D9, 13B1, 14D5, 10F10, 13C9, 10C6 and 13D3) to be similar to the MAbs reported by Salfeld et al. (1989) which had recognition sites around residue 140 and for which the epitope was termed HBe2. We define HBefl and the HBe2 regions as two domains structurally separated by the proline at position 134 in the HBeAg sequence, by using a panel of murine MAbs and human sera. The recognition sites were mapped to a single amino acid resolution. Mapping data obtained by the use of recombinant proteins and synthetic peptides were found to corroborate each other. Our findings resolve earlier observed discrepancies with respect to the immune recognition of the C-terminal part of the HBeAg (Salfeld et al., 1989; S~tllberg et al., 1991 b;

1339

Bichko et al., 1992). The C-terminal part of the HBeAg, residues 121 to 149, induces MAbs that recognize overlapping epitopes, which is the most likely reason for the variation in the previous observations. Also, in the different reports, varying selection criteria for the primary hybridomas have been used, such as specific inhibition of human anti-HBe (Imai et al., 1982; Ferns & Tedder, 1984; Noah & Harthus, 1987), or direct binding to rHBeAg or drHBcAg (P. Sillekens et al., unpublished; Bichko et al., 1992). Taken together, our data would suggest that the main surface-exposed part of the C terminus of the native HBeAg is the region 124 to 133, whereas the region 134 to 146 in most cases seems to be inaccessible to antibodies. Evidence for this is that (i) all HBefl-specific MAbs recognizing residues 128 to 133 can capture recombinant and native HBeAg, whereas the other MAbs can not, and (ii) the HBefl-specific MAbs recognizing residues 128 to 133 compete with human antibodies in commercial assays, whereas the MAbs raised against rHBeAg and drHBcAg, recognizing sites only two residues closer to the C terminus, do not. In structural terms, this is possibly due to a predicted turn induced by the two prolines at residues 134 and 135 (Chou & Fasman, 1978), which would make the Cterminal part 136 to 149 internal and thereby less accessible to antibodies. We gratefully acknowledge Dr H. P. Harthus, Mrs K. Rot and Mrs S. van Meerten for skilful help in the production of the MAbs.

References ALTER, H. J., SEEFF, L. B., KAPLAN, P. M., McAULIFVE,J., WRIGHT, E. C., GERIN, J. L, PURCELL,R. H., HOLLAND,P. V. & ZIMMERMAN, H. J. (1976). The infectivity of blood positive for e antigen and DNA polymerase after accidental needlestick exposure. New England Journal of Medicine 295, 909. BICHKO, V. V., PUSHKO,P., DREILINA,D. D., PUMPEN,P. & GREN, E. (1985). Subtype ayw variant of hepatitis B virus: DNA primary structure analysis. FEBS Letters 185, 208~12. BICHKO, V., V., SCHODEL, F., NASSAL, M., GRENS, E., BERZINSH, I., BORISOVA, G., MISKA, S., PETERSON,D. L., GREN, E. & WILL, H. (1992). Epitopes recognized by antibodies to denatured core protein of hepatitis B virus. Molecular Immunology 30, 221-231. BONINO, F., BRImETTO, M.R., R1ZETTO, M. & WILL, H. (1991). Hepatitis B virus unable to secrete ' e' antigen. Gastroenterology 100, 1138 1141. BRUSS,V. & GERLICH,W. H. (1988). Formation of transmembraneous hepatitis Be-antigen by cotranslational in vitro processing of the viral precore protein. Virology 163, 268-275. BUDKOWSKA, A., KALINOWSKA, B. & NOWOSLAWSKI, A. (1979). Identification of two HBeAg subspecificifies revealed by chemical treatment and enzymatic digestion of liver-derived HBcAg. Journal of Immunology 123, 1415 1416. CHOU, P.Y. & FASMAN, G.D. (1978). Prediction of the secondary structure of proteins from their amino acid sequence. Advances in Enzymology 47, 45-148. FERNS, R.B. & TEDDER, R.S. (1984). Monoclonal antibodies to hepatitis Be antigen (HBeAg) derived from hepatitis B core antigen (HBcAg): their use in characterization and detection of HBeAg. Journal of General Virology 65, 899-908.

1340

M . Siillberg and others

HENIKOFF, S. (1984). Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28, 351-353. IMAI, M., NOMURA, M., GOTANDA, T., SANO, T., TACHIBANA,K., MIYAMOTO, H., TAKAHASHI,K., TOYAMA, S., MIYAKAWA, Y. & MAYUMI,M. (1982). Demonstration of two determinants on hepatitis B e antigen by monoclonal antibodies. Journal of Immunology 128, 69-72. KOZLOVSKAYA,T. M., PUMPEN,P. P., DREILINA,n. n., TSIMANIS,A. J., OSE, V. P., TSIBINOGIN,V. V. & GREN, E.J. (1986). Formation of capsid-like structures as a result of expression of cloned coat protein gene of RNA bacteriophage fr. Doklady Akademii Nauk U.S.S.R. (in Russian) 287, 452-455. LAEMMLI, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, London 227, 680-685. MAGNIUS, L. O. & ESPMARI¢,A. (1972). New specificities in Australia antigen positive sera distinct from the Le Bouvier determinants. Journal of Immunology 109, 1017-1021. MILICH, D. R., JONES, J., HUGHES, J. L., PRICE, J., RANEY, A. K. & MCLACnLAN, A. (1990). ls a function of a secreted hepatitis B e antigen to induce tolerance in utero? Proceedings of the National Academy of Sciences, U.S.A. 87, 6599-6603. NOAH, M. & HARTHUS, H. P. (1987). Immunogenicity of hepatitis B core antigen: monoclonal (mouse) and polyclonal (human) anti-HBc antibodies recognizes the same epitope. Zentralblattfiir Bakteriologie und Mikrobiologie 267, 94. OKADA, K., KAMIYAMA,I., INOMATA,M., IMAI,M., MIYAKAWA,M. & MYUMI, M. (1976). 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, 749. ONO, Y., ORADA, H., SASADA, R., IGARASHI, K., SUGINO, Y. & NISHIOKA,K. (1983). The complete nucleotide sequence of the cloned hepatitis B virus DNA: subtypes adr and adw. Nucleic Acids Research 11, 1747-1757. Ou, J., YEll, C. & YEN, T. (1986). Hepatitis B virus gene function: the precore region targets the core antigen to cellular membranes and causes the secretion of the e antigen. Proceedings of the National Academy of Sciences, U.S.A. 83, 1578 1582. SALEELD,J., PFAFF, E., NOAH, M. & SCHALLEe,,H. (1989). Antigenic determinants and functional domains in core antigen and e antigen from hepatitis B virus. Journal of Virology 63, 798-808.

S.&LLBERG,M., NORDER, H., WEILAND,0. & MAGNIUS,L. O. (1989). Immunoglobulin isotypes of anti-HBc and anti-HBe, and hepatitis B virus (HBV) DNA elimination in acute hepatitis B. Journal of Medical Virology 29, 296-302. SALLBERG,M., RUDdy, U., MAGNIUS,L. O., HARTHUS,H. P., NOAH, M. & WArmEN, B. (1991 a). Characterization of a linear binding site for a monoclonal antibody to hepatitis B core antigen. Journal of Medical Virology 33, 248-252. SY,LLBERG,M., RUD~N, U., WAHREN,B., NOAH, M. & MAGNIUS,L. O. (1991 b). Human and routine B-cells recognize the HBeAg/beta (or HBe2) epitope as a linear determinant. Molecular Immunology 28, 719~26. S~LLBERG,M., RUDI~N,U., MAGNIUS,L. O., NORRBY,E. & Wm~REN, B. (1991 c). Rapid 'tea-bag' peptide synthesis using fluorenylmethoxycarbonyl (Fmoc) protected amino acids applied for antigenic mapping of viral proteins. Immunology Letters 30, 59-68. SANGUR,F., NICKLEN, S. & COULSON,A. R. (1977). DNA sequencing with chain-terminating inhibitors of the National Academy of Sciences, U.S.A. 74, 5463-5467. STANDING, D., Ou, J., MASlARZ, F. & RUTTER, W. (1988). A signal peptide encoded within the precore region of hepatitis B virus directs the section of a heterogeneous population of e antigens in Xenopus oocytes. Proceedings of the National Academy of Sciences, U.S.A. 85, 8405 8409. TAKAHASHI,K., MACH1DA,A., FUNATSU,G., NOMURA,M., USUDA,S., AOYAGI, S., TACHIBANA,K., MIYAMOTO,H., IMAI, M., NAKAMURA, T., MIVAKAWA,Y. & MAVUMI,M. (1983). Immunochemical structure of hepatitis B e antigen in the serum. Journal of Immunology 130, 2903-2907. TAKAHASHI,K., KISH1MOTO,S., OHIRO,K., YOSHIZAWA,H., MACHIDA, A., OHNUMA, H., TSUDA, F., MUNEKATA,E., MIAYKAWA,Y. & MAYUMI, M. (1992). Molecular heterogeneity of e antigen polypeptides in sera from carriers of hepatitis B virus. Journal of Immunology 147, 3156-3160. TOWBIN, H., STAEHEEIN,T. & GORDON, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences, U.S.A. 76, 4350-4354. UY, A., BRuss, V., GERLICH,W. H., KOCHEL, H. G. & THOMSSEN,R. (1986). Precore sequence of hepatitis B virus inducing e antigen and membrane association of the viral core protein. Virology 155, 8946.

(Received 24 November 1992; Accepted 24 February 1993)