Institute of Medical Virology1, and Institute of Biochemistry4, Clinics of the Justus-Liebig-University, Frankfurter Str. 107, D-35392 Giessen, Germany
Institute of Biomedical Research, Riga, Latvia2
Institute of Virology, Humboldt University, D-10098 Berlin, Germany3
Institute of Medical Microbiology, Georg-August University, D-37075 Göttingen, Germany4
Author for correspondence: Wolfram H. Gerlich. Fax +49 641 99 41209.e-mail Wolfram.H.Gerlich{at}viro.med.uni-giessen.de
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Abstract |
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Introduction |
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The MHBs protein has a 55 aa N-terminal extension named the preS2 domain and an S domain which is identical to SHBs. The preS2 domain is hydrophilic and does not contain cysteines. Asn-4 is always glycosylated with a complex biantennary glycan which leads to MHBs glycoproteins GP33 and GP36 (Schmitt et al., 1999 ). The preS2 domain is located at the surface and may partially cover the S domain of MHBs (Heermann et al., 1984
). Peptides with the N-terminal half of preS2 were found to induce protective immunity (Itoh et al., 1986
; Neurath et al., 1986a
; Neurath & Kent, 1988
). The preS2 domain binds to polymerized human serum albumin (pHSA) (Machida et al., 1984
; Krone et al., 1990
), but the binding site has not yet been accurately mapped. The LHBs protein contains the three domains preS1, preS2 and S (Heermann et al., 1984
). The preS1 sequence 2147 is involved in binding of the virus to hepatocytes and in the induction of neutralizing antibodies (Neurath et al., 1986b
). LHBs is much more abundant in HBV particles and HBs filaments than in HBs spheres. Due to its biosynthesis, Asn-4 of the preS2 domain is not glycosylated in LHBs (Bruss et al., 1994
).
The preS2 sequence, as part of MHBs/SHBs mixed HBs particles, is able to induce an antibody response against SHBs in mice which are non-responsive to SHBs alone (Milich et al., 1985 ; Milich, 1997
). Non-responsiveness to SHBs is a major problem in certain groups of human recipients (Miskovsky et al., 1991
). Furthermore, HBV escape mutants with mutations in the major SHBs epitopes exist (Carman et al., 1989
). Thus, several alternative HBV vaccines with S, preS2 and preS1 sequences have been developed (Shouval et al., 1994
; Thoma et al., 1990
; Couillin et al., 1999
; Janowicz et al., 1991
). While these vaccines induce good anti-SHBs responses in humans and readily detectable anti-preS2 responses in animals, the anti-preS2 responses in humans seem, for unknown reasons, to be weak (W. Gerlich, unpublished). Potential reasons for these results may be unexpected variable modifications of the preS2 such as O-glycosylation (Tolle et al., 1998
; Schmitt et al., 1999
; Janowicz et al., 1991
; Prange & Streeck, 1995
) or terminal acetylation (Schmitt et al., 1999
) and subtype or genotype differences which alter the immunogenicity of the vaccines and the recognition of anti-preS2 antibodies. Therefore, we wanted to characterize the B-cell epitopes of natural HBsAg as they would be present during natural infection. In this respect, the interference of modified HSA with the accessibility of epitopes is of special interest and was also studied.
We (Meisel et al., 1994 ) and others (Howard et al., 1991
; Neurath et al., 1986c
; Milich et al., 1985
; Mimms et al., 1990
) have previously mapped several mouse-derived MAbs against the preS2 domain of natural HBsAg particles, but the immunization and selection procedure favoured generation of MAbs against group-specific epitopes (Meisel et al., 1994
) or was not well-specified in this respect. Here, we used HBs spheres from one HBV carrier with a well-defined protein composition (Schmitt et al., 1999
) for immunization. We analysed the domain and the genotype specificities of the isolated MAbs using antigens from the same and from different HBV genotypes and, in addition, recombinant proteins and peptides. We could assign some preS2 MAbs to the previously defined epitope groups I to III, but the immunodominant preS2 region identified in our study did not map to these groups. The new MAbs may be used for testing the quality of HBsAg preparations and for fine analysis of the immune response against HBs proteins in recipients of preS-containing vaccines.
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Methods |
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Peptides.
The peptides covering preS2 sequence 126 ayw2, adw2, 111 D, ayw2, 112 ayw2 and 1122 ayw2 were kindly provided by M. Bienert (Humboldt University, Berlin, Germany). PreS2 peptides 825 ayw2, adw2 and 1330 adw were synthesized by P. Pushko and A. Tsimanis (Institute of Biomedical Research, Riga, Latvia) by the tea-bag solid-phase method using FMOC-protected amino acids (Bachem). Peptides were dissolved in water to a concentration of 2 mg/ml without further purification. Tryptic peptides from the preS2 domain of natural HBsAg/ayw2 particles were prepared as described by Schmitt et al. (1999) .
Enzyme immunoassay (EIA).
Polysorb microplates with flat bottoms (Nunc) were incubated with 100 ng highly purified HBsAg in 100 µl PBS per well for 1220 h at 4 °C. After removal of the liquid, 200 µl of 1% BSA/PBS was added to each well for 2 h at 37 °C. Synthetic peptides were coated onto Nunc maxisorb microplates at 10 µg/ml in 0·005 M sodium phosphate buffer, pH 9·6. Tryptic peptides from natural HBsAg particles were diluted 1:50 in PBS and coated onto Nunc polysorb plates. Washing was usually done twice with 0·25% Tween 20/PBS and three times with PBS; for synthetic peptides, 0·05% Tween 20 in PBS was used. Hybridoma supernatants (100 µl) containing RPMI-1640 medium with 10% foetal calf serum were added to the wells and incubated for 1 h at room temperature. If appropriate, dilutions were made in 0·1% BSA/PBS. After five washings as before, 100 µl peroxidase (POD)-labelled antibody against mouse IgG (affinity-purified from goat or rabbit serum; both from Sigma) diluted 1:1000 in 0·1% BSA/PBS was added to each well for 1 h at room temperature. After five washings, as described above, an o-phenylenediamine/H2O2 substrate (tablets from Abbott Laboratories) was added for 15 min at room temperature and the amount of coloured product was measured by A492.
Recombinant preS2 proteins.
For WB, we used frCP-preS2 expression protein libraries constructed on the basis of plasmid pFRd8 and its derivatives. As a source of preS2 fragments, we used plasmids pHB320 and PAE10 containing cloned HBV genomes subtypes ayw2 (Bichko et al., 1985 ) and adw2 (Meisel et al., 1993
), respectively. Fragments of different size were cloned within polylinkers inserted into the frCP gene. Libraries of recombinant frCP-preS2 coding plasmids were expressed in Escherichia coli K802 cells. Bacteria were pelleted, suspended in SDSPAGE electrophoresis buffer containing 2% SDS and 2%
-mercaptoethanol, and lysed at 100 °C for 5 min.
Western blotting (WB).
SDSPAGE was done by standard procedures using a Tall-Mighty-Small chamber (Hoefer). The stacking and separation gels contained 3·25% and 12·5% polyacrylamide, respectively. Purified HBsAg (50 µg) in 50 µl solution was mixed with 5x Laemmlis sample buffer and stacking buffer to yield 1% SDS/DTT in 250 µl. Samples were then boiled for 5 min and subjected to electrophoresis in one wide slot for 1·5 h at 100150 V until bromophenol blue was close to the lower edge of the gel. The separated proteins were blotted onto a PVDF membrane (Millipore) in a semi-dry transversal electroblotting chamber at 8·8 mA/cm2 for 1 h. Every run included one narrow slot with protein markers (Bio-Rad low range). The strip with the protein marker was cut off and stained with Coomassie blue; the membrane with the separated HBs proteins was shaken overnight with 5% milk powder in PBS at 4 °C (Gültekin & Heermann, 1988 ). After three washings with 0·25% Tween 20/PBS, the membrane was cut into 4 mm wide strips. The strips were incubated with 200 µl antibody solution and 25 µl 10% BSA/PBS for 2 h at room temperature. After washing three times with 0·05% Tween 20/PBS, the strips were incubated with 500 µl anti-mouse IgG F(ab)' fragment conjugated with alkaline phosphatase (Boehringer Mannheim) diluted 1:1000 in 0·1% BSA/PBS. Bound conjugate was detected either with NBT/BCIP substrate (Sigma) or by ECL (Amersham). Reference MAbs were: MA18/7 for LHBs (Heermann et al., 1984
); Q19/10 for MHBs (Heermann et al., 1988
); and H166 for SHBs (Peterson et al., 1984
). WB for frCP-preS2 fusion protein was done as described previously (Meisel et al., 1994
). The WB shown in Fig. 5
were not cut into strips and bound antibody was revealed using POD-labelled anti-mouse antibody (Dianova) diluted 1:2000 in 0·1% BSA/PBS and ECL.
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Binding of pHSA and inhibition of binding to HBsAg.
Microplates were coated with HBsAg as described before and post-coated with foetal calf serum for 2 h at 37 °C. Fifty microlitre samples of MAbs or dilutions thereof were mixed with 50 µl 1:100 pHSAPOD oligomers in 0·1% BSA/PBS. The mixture was transferred to the HBsAg-coated microplates and incubated for 1·5 h at room temperature. The plates were washed as before and bound POD was detected also as before. Positive controls for inhibitory activity contained unlabelled pHSA in 1:2 dilution and a well-studied preS2-specific MAb S26 (ascites diluted 1:1000; described by Sominskaya et al., 1992 ), which inhibits pHSA binding to preS2 very strongly. Each run included at least four negative controls with BSA/PBS which yielded absorbance values around 3. Values below 50% of the negative controls were considered positive. Very strong inhibition yielded absorbance values below 0·1.
Tryptic cleavage of preS2 protein.
For partial digestion of HBsAg at the solid phase of microplates, 1 µg trypsin (Boehringer Mannheim) in 100 µl TrisHCl, pH 8·0, was added to each coated well for various time periods, removed and the reaction was stopped by addition of the trypsin inhibitor Pefabloc (Boehringer Mannheim). Thereafter, EIA was carried out with the various MAbs as described above. By this treatment, the N-terminal fragments are stepwise removed and epitopes on these fragments can no longer be detected. For WB of partial digests each 0·6 µg HBsAg/ayw2 was mixed with 0·1 µg trypsin and incubated for 1, 5, 10, 15, 20, 40 or 60 min. The reaction was stopped by adding SDSPAGE buffer containing 2% SDS and 10% DTT and immediately denaturing the sample at 95 °C for 10 min.
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Results |
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PreS2-specific MAbs
The remaining seven MAbs were preS2-specific according to WB (Fig. 1b; Table 1
). They reacted with MHBs and partially with LHBs, but not with SHBs. Epitopes of MAbs 1-8H1 and 1-8H2 seemed to require the glycoside bound to Asn-4 of MHBs due to the fact that only GP33 and GP36, but no LHBs could be detected in WB. Removal of the glycan with PNGase F led to a complete loss of reactivity of MAb 1-8H1 in EIA (data not shown) as was previously described for MAb Q19/10 (Heermann et al., 1988
).
MAbs 2-11B1, 3-11C1 and 3-11C2 were similar to each other in that they reacted strongly with HBsAg/ayw2, but reacted only very weakly or not at all with HBsAg/adw2.
MAb 2-12F2 seems to be universally reactive with preS2 both in EIA and WB and with both HBsAg genotypes used here. MAb 1-9D1 reacted in EIA very well with HBsAg/ayw2 and rather weakly with HBsAg/adw2. In WB, a moderately strong reaction was only found with HBsAg/ayw2 showing MHBs and LHBs.
Mapping of preS2-epitopes
PreS2-specific MAbs have been divided into three subgroups (see Fig. 2) as suggested by Mimms et al. (1990)
and by Meisel et al. (1994)
. Group II antibodies, according to Mimms et al. (1990)
, bind to epitopes at the very N-terminal-most region of MHBs. Such antibodies react in WB only with MHBs where the Asn-4 of preS2 is glycosylated, but not with LHBs where Asn-4 of preS2 is not glycosylated. Reference MAb Q19/10 is such an antibody. A similar pattern is shown by our MAbs 1-8H1 and 1-8H2. Surprisingly, these antibodies show, in contrast to Q19/10, a stronger reactivity with HBsAg/ayw2 than with HBsAg/adw2 in EIA (data not shown) and in WB (Fig. 1b
). HBsAg/ayw3 expressed in C127 mouse cell line (Hepagene) did not react at all with MAb 1-8H1 for unknown reasons.
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Detection of the larger MHBs fragments generated by trypsin in WB using either MAb 2-12F2 or 1-9D1 confirmed these results. A protein fragment containing the epitope of MAb 2-12F2 appeared after 1 min digestion in the liquid phase indicating cleavage at position Arg-16 and the epitope disappeared within 40 min, due to complete cleavage at Arg-18 (Fig. 5). In comparison, the protein fragment containing the epitope of MAb 1-9D1 also appeared after 1 min digestion but then remained stable for 60 min indicating slow cleavage at Arg-48.
Using the HPLC-purified preS2 fragments from complete tryptic digestion of HBsAg/ayw2, MAbs 2-11B1, 3-11C1 and 3-11C2 bound to fragment 116, and MAb 1-9D1 bound to fragment 1948 in EIA. MAb 2-12F2 did not react with any fragment due to the fact that its epitope contains Arg-18.
The binding sites of MAbs 2-11B1, 3-11C2 and 2-12F2 were also fine-mapped using recombinant frCP-preS2 proteins in WB (Table 2. The epitopes of MAbs 3-11C2 and 2-12F2 were further narrowed down by using various synthetic peptides as shown in Table 3
. The data contributed to the mapping as shown in Fig. 2
. A reaction was also obtained for MAb 1-9D1 with frCP-preS2 (148) genotype D. The reaction can only be due to sequence 3648 because sequence 1935 is identical in HBsAg/ayw2 and ayw3. Since MAb 1-9D1 bound to the O-glycosylated and non-glycosylated tryptic preS2 fragment 1948 from natural HBsAg, it is suggested that Thr-37, which is conjugated with an O-glycan, is not part of the epitope. Thus, the epitope of 1-9D1 is narrowed down to residues 3848 which carry the subtype-specific group III epitopes. Phe-46 is part of the epitope since digestion of HBsAg with chymotrypsin gave rise to a stable MHBs intermediate at this cleavage site which was no longer reactive in EIA (data not shown).
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Discussion |
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The relatively greater abundance of preS2-specific MAbs in our study is consistent with the conclusion of Neurath et al. (1984) that the preS2 domain may be immunodominant in BALB/c mice with MHC genotype H2d. Within the preS2 sequence, two overlapping sequences, 1420 and 1824, were previously described as immunodominant and subtype-independent (Milich et al., 1987
; Milich, 1997
). Our own previous study on preS2-specific MAbs (Meisel et al., 1994
) seemed to confirm these reports. However, the mice used for generation of these MAbs were immunized with one genotype and boostered with another, preferably stimulating group-specific antibody production. Using the same genotype for priming and boosting, the present study generated predominantly genotype-specific preS2 MAbs, some of which even distinguished genosubtypes. We found only one of seven MAbs (2-12F2) which belonged to group I and had an epitope located within the so-called immunodominant conserved preS2 sequence, aa 1424. All the other epitopes, overlapping with this region, reached from position 315 and were genotype D-specific. We suggest epitopes ranging from aa 315 and 1324 to distinguish groups IA and IB, respectively.
The only known function of the sequence covering group IA epitopes is the binding of HSA. The binding is species-specific and does not occur with non-primate serum albumin (Machida et al., 1984 ). The binding is strongly enhanced by polymerization of albumin with glutardialdehyde but not with other reagents (Yu et al., 1985
). Polymerization with glutardialdehyde possibly mimics a naturally occurring modification because a subfraction of HSA also binds to preS2 (Krone et al., 1990
). This albumin dissociates from preS2 during purification in high molarities of salt as used in this study. Thus, the albumin-binding sites in preS2 were fully accessible to the antigens used for immunization in this study and can stimulate antibody production against epitopes within the albumin-binding site. Our findings suggest that the albumin-binding site is preferentially recognized by the B-cells of BALB/c mice. Possibly, these epitopes can also be recognized in human recipients of preS2-containing HBsAg vaccines. Previous studies have shown that 10 µg/ml MHBs-containing HBs particles are sufficient to exhaust the fraction of serum albumin which binds to preS2 (Krone et al., 1990
). However, in vaccine recipients, 1040 µg HBsAg encounter ca. 3000 ml human serum. The binding site of modified HSA seems to be completely within sequence 316, because neither glycoside-dependent group II MAbs 1-8H1 and Q19/10 nor the group IB MAb 2-12F2 significantly inhibited pHSA binding. Genotype-specific preS2 MAbs like 1-9D1 have been previously described as group III (Milich et al., 1985
; Mimms et al., 1990
; Meisel et al., 1995). Both in our previous work and in this study, only one of six or seven preS2 MAbs belonged to this group. In agreement with the previous mapping of group III MAbs, the epitope of MAb 1-9D1 is between aa 3848. Positions 3554 of preS2 are most variable between and even within HBV genotypes. This suggests that this region may be under heavy pressure from the immune system. The relatively low number of MAbs against this region would, however, mean that it is not immunodominant in mice, possibly because it is less flexible than region 523. This presumption is consistent with the rapid cleavage of MHBs by trypsin at Arg-16 and Arg-18 but the slow cleavage at Arg-48. Nevertheless, region 3554 may be the more accessible B-cell target in humans because region 315 may be covered by HSA. Another masking of preS2 epitopes occurs at Thr-37 of MHBs genotype D in natural HBsAg by O-glycosylation (Schmitt et al., 1999
). In fact, preS2 epitopes involving Thr-37 have not yet been described. The reason that the highly hydrophilic and highly conserved sequence 2434 has also not yet been identified as a target for antibodies remains unknown.
In summary, the preS2 domain of natural HBsAg particles is immunodominant in mice in spite of being a minor polypeptide component. However, its immunodominant group IA epitopes overlap with a binding site for HSA and may thus, not be available in humans. This could explain the weak and transient anti-preS2 response in most HBV patients or recipients of preS2-containing vaccines. Other potential epitopes of preS2 are less accessible, modified by glycans and/or highly variable. This makes recognition of antibodies against these other regions difficult and unreliable. While preS2 would still probably be a valuable addition to HBV vaccines due to its T-cell epitopes (Cupps et al., 1993 ), the evaluation of such vaccines may be more complex than anticipated. The predominantly subtype-specific antibody response against both SHBs and preS2 epitopes suggests that HBV vaccines containing more than one genotype may be useful. It should be noted that the first HBV escape mutant was found in recipients of a vaccine containing SHBs genotype A while the escape virus had genotype D (Carman et al., 1989
).
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Acknowledgments |
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References |
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Received 3 August 1999;
accepted 28 September 1999.