Departments of Clinical Virology1 and Dermatovenereology2, Göteborg University, Guldhedsgatan 10 B, S-413 46, Göteborg, Sweden
Author for correspondence: Tomas Bergström. Fax +46 31 827032. e-mail Tomas.Bergstrom{at}microbio.gu.se
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Abstract |
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Introduction |
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The approximately 50% base sequence identity between HSV-1 and HSV-2 has hampered attempts to differentiate between infections with the two subtypes by serological means (Ashley et al., 1991 ; Field et al., 1993
; Mertz, 1990
). Each of the two viruses expresses at least 10 envelope glycoproteins but, of these, the only glycoproteins that induce an exclusively type-specific B-cell response are glycoproteins G from HSV-1 and HSV-2 (gG-1 and gG-2, respectively). Therefore gG-1, a 238 amino acid envelope protein of unknown function (McGeoch et al., 1985
), has been suggested as a prototype antigen for type-specific HSV-1 serology (Lee et al., 1986
; Sanchez-Martinez et al., 1991
) and was recently described in use, in a pre-market evaluation of a commercial enzyme immunoassay, by Ashley et al. (1998)
. Its counterpart on HSV-2, gG-2, has also been used successfully as antigen in type-specific HSV-2 seroassays (Ho et al., 1992
; Lee et al., 1985
; Svennerholm et al., 1984
). Each of these two glycoproteins contains relatively long stretches of type-unique amino acids, believed in the past to harbour the type-specific epitopes. However, recent investigations have shown that human linear B-cell epitopes on gG-2 are mainly localized to the gG-1-homologous stretches of the protein (Grabowska et al., 1999
; Liljeqvist et al., 1998
; Marsden et al., 1998
).
In the present study, we have exploited the pepscan technique (Frank, 1992 ) to attempt to localize the epitopes on gG-1, by using purified anti-gG-1 antibodies from HSV-1-isolation-positive patients, a method that has previously been used for mapping both linear (Kramer et al., 1994
) and discontinuous (Gao & Esnouf, 1996
; Reineke et al., 1996
) epitopes. In contrast to the narrow IgG-reactive stretches found on gG-2 (Liljeqvist et al., 1998
), the epitopes on gG-1 were multiple and distributed widely over the protein, excluding the gG-1-unique sequence at the amino terminus. However, one immunodominant domain was identified and peptides representing this region reacted with all the purified anti-gG-1 antibodies investigated.
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Methods |
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The virus strains used for antigen production for different assays were HSV-1 (F) for type-common ELISA (Jeansson et al., 1983 ) and HSV-2 strain B4327UR (isolated by S. Jeansson at Göteborg University), a local wild-type strain, for detection of IgM antibodies, as well as type 2-specific IgG antibodies. A randomly selected clinical HSV-1 isolate (VI 951058) was employed to produce antigen for immunoblot strips. Patient isolates were taken primarily from oral lesions, but also from vulva, penis, finger, nose and neck. HSV was isolated on GMK-AH1 cells and subtyped as HSV-1 by an assay using a type-specific monoclonal antibody (MAb), designated B1.C1.B4, directed against glycoprotein C-1, as described previously (Bergström et al., 1992
; Nilheden et al., 1983
).
Serum samples and MAbs.
Sera were collected from 21 HSV-1-isolation-proven patients and stored frozen at -20 °C. A commercially available anti-gG-1 mouse MAb, designated 13-122-100 (Advanced Biotechnologies), was used in immunoblots and epitope-mapped by a pepscan method (see below). Human sera were first characterized by an ELISA based on a type-common HSV antigen and, if positive, were tested for the presence of HSV-1- and HSV-2-specific antibodies. The HSV status was finally confirmed by the Western blot (immunoblot) technique.
Purification of anti-gG-1 antibodies from HSV-1-positive sera.
A recombinant gG-1 antigen, truncated at the transmembrane region and prepared in CHO cells, was kindly provided by SmithKline Beecham Biologicals. The gG-1 antigen (1 mg antigen/ml gel) was coupled to CNBr-activated Sepharose 4B (Pharmacia Biotech) according to the manufacturers instructions. Sera from the HSV-1-isolation-proven patients were diluted 1:5 in Tris-buffered saline and 0·5% Tween 80, applied and recirculated through the column for 1 h. The unbound fraction was retained and later evaluated in an ELISA. Elution of the antibodies was achieved by the addition of 0·1 M glycineHCl (pH 2·8) as described previously (Liljeqvist et al., 1998 ).
ELISA.
The type-common ELISA was based on sodium deoxycholate-solubilized membranes from HSV-infected cells (Jeansson et al., 1983 ). The IgG response to HSV-2 was characterized by using Helix pomatia lectin-purified gG-2 as the HSV-2-specific antigen (Olofsson et al., 1986
; Svennerholm et al., 1984
). The HSV-1-specific test was based on gG-1 and analysed according to an ELISA protocol from SmithKline Beecham as follows: the gG-1 antigen (180 µg/ml) was diluted 400-fold in PBS and coated on Maxisorp F96 immunoplate microplates (Nunc) overnight at 4 °C. Sera were diluted in 2-fold steps from 1:100 to 1:6400, purified anti-gG-1 antibodies from 1:25 to 1:400. PBS was used as the diluent and supplemented with 1% BSA and 0·05% Tween 20. Alkaline phosphatase-conjugated, affinity-purified F(ab')2 fragment goat anti-human IgG (Jackson ImmunoResearch) was used as a conjugate, at a 1:3500 dilution, with Sigma 104 phosphatase substrate tablets (Sigma) in diethanolamine buffer (pH 9·8) as a substrate. The microplates were read in a Vmax kinetic microplate reader (Molecular Devices) at 405 nm with 650 nm as the reference wavelength. The mean absorbance of 20 negative sera to the gG-1 antigen was 0·143 (SD=0·05) and the cut-off level for the HSV-1 type-specific assay, as well as for the type-common ELISA, was defined as the mean absorbance plus 0·2 absorbance units. For the HSV-2 type-specific assay, the cut-off level was defined as the reactivity of a standard high-titrating HSV-1 positive serum (0·390) plus 0·1 absorbance units (Svennerholm et al., 1984
).
Indirect immunofluorescence.
An in-house test was employed for the detection of type-common HSV IgM-antibodies, based on acetone-fixed HSV-2-infected (strain B4327UR) GMK-AH1 cells. Fifty µl sera was added to 50 µl PBS and then titrated in 2-fold steps in a serial dilution. Fluorescein-labelled goat anti-human IgM liquid globulin (bioMérieux) was used as the conjugate. The samples were analysed in an immunofluorescence microscope (Nikon HB10101AF).
Immunoblotting.
Antigen for immunoblot strips was produced by infecting HEp-2 cells with a clinical HSV-1 isolate (VI 95-1058). Infected cells were sonicated and subjected to SDSPAGE under reducing conditions. The proteins were then electrotransferred to nitrocellulose membranes (Towbin et al., 1979 ). Immunoblotting was also performed on strips prepared in a mini-blot system (XCell II Mini Cell System; Novex) assayed as described above. Strips were incubated overnight with HSV-positive and HSV-negative sera at a 1:50 dilution, purified anti-gG-1 antibodies at 1:5 and the anti-gG-1 MAb at 1:50. Peroxidase-labelled anti-human and anti-mouse IgG antibodies (Dako) diluted 1:100 were used as the conjugate and 4-chloro-1-naphthol (Bio-Rad) as the substrate.
Pepscan analysis.
A panel of 13-mer peptides with 10 overlapping amino acids, covering the entire gG-1 sequence, was prepared on a continuous cellulose-membrane support (Jerini Bio Tools) according to a standard spot-synthesis protocol (Frank, 1992 ). The sequence was based on data from McGeoch et al. (1985)
and the investigation of HSV-1 strain 17. Initially, purified anti-gG-1 antibodies from one of the HSV-1-positive sera were diluted in blocking buffer (Genosys Biotechnologies) and tested for reactivity. Next, purified anti-gG-1 antibodies from the remaining 20 HSV-1-positive sera were pooled in two lots (10 sera in each) and incubated with the membrane at a dilution correlated to their gG-1 antigen-based ELISA titres. To define the epitopes for purified anti-gG-1 antibodies from 10 of the pooled serum samples more closely, new cellulose membranes were synthesized containing all reactive peptides and each sample was incubated separately at a 1:30 dilution. For fine-mapping of the anti-gG-1 MAb, 15-mer peptides with 14 overlapping amino acids were used as described above, diluted in blocking buffer and incubated at a concentration of 0·1 µg/ml. Antibody binding was detected with ECL detection reagent solution (Amersham) after addition of peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch) at a concentration of 0·2 µg/ml, as described previously (Liljeqvist et al., 1998
). For re-use, membranes were regenerated according to the manufacturers protocol with a regeneration buffer containing 50 mM TrisHCl, pH 6·7, 100 mM 2-mercaptoethanol and 2% SDS. Before re-use, the membrane was incubated as described above with the conjugate alone to exclude residual activity. The demarcation of the reactive stretches was based on visual assessment, in which dark spots represented binding between the antibodies and the peptide.
Competitive indirect ELISA of purified human anti-gG-1 antibodies.
To investigate whether binding of purified anti-gG-1 antibodies could be blocked by peptides, we selected anti-gG-1 antibodies from four samples that were mapped to two to four epitopes each (data derived from the pepscan analysis). Synthetic peptides (synthesized by KJ Ross-Peterson A/S) comprising the suggested epitopes were incubated at different concentrations (1 ng/ml to 20 µg/ml) with the purified anti-gG-1 antibodies (diluted 1:101:50) for 18 h at room temperature to allow binding. The mixtures were then assayed in an ELISA, with gG-1 as the antigen, as described above. gG-2 peptides, at similar concentrations, were used as controls. A synthetic peptide comprising the predicted epitope for the anti-gG-1 MAb was incubated at concentrations ranging from 1 ng/ml to 10 µg/ml, as described above, with the MAb at a dilution of 1:800. Inhibition was evaluated by using the gG-1 antigen in an indirect ELISA.
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Results |
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Results from immunoblots of sera are shown in Table 1. Sera were defined as HSV-1-positive if gB-1, gD-1, gC-1 and/or gG-1 were visible and HSV-2-positive if gB-2, gD-2 and gG-2 were identified. Reactivity of the anti-gG-1 MAb, as well as the purified anti-gG-1 antibodies, in immunoblots was shown in the form of two sets of bands, one identifying the gG-1 protein as having an apparent molecular mass of 3841 kDa, the other with a lower intensity and an apparent molecular mass of 4453 kDa (data not shown). According to Lee et al. (1986)
, these bands probably represent components of gG-1 at different stages of maturation. The negative-control sera were nonreactive when analysed by immunoblot (data not shown).
Pepscan analysis of purified anti-gG-1 antibodies
In order to define the gG-1 epitopes, overlapping peptides covering the entire gG-1 sequence were coupled to a cellulose membrane. Initially, purified anti-gG-1 antibodies from one patient (patient no. 9) were evaluated (Fig. 1) and, after that, sera from 10 patients were pooled and mapped. All peptides reactive in this experiment were then synthesized as 13-mer peptides and each sample of the purified anti-gG-1 antibodies was incubated separately. Multiple epitopes were detected for each of the 11 more-closely investigated samples and the reactive spots were mainly localized to the central portion of the glycoprotein. The number of reactive stretches for the different patient sera varied between two and five. An immunodominant region, delimited by amino acids 112127 and located in a region with a high degree of similarity to gG-2, was reactive to all 11 sera. To minimize the risk of omitting possible epitopes due to the small number of sera tested, an additional 10 purified sera were pooled and tested for binding to the gG-1 membrane, showing reactivity to the same regions but also to one additional domain situated in the amino terminus, non-homologous to gG-2. The epitope of the anti-gG-1 MAb was mapped to amino acids 110AFPL113, adjacent to the human immunodominant region. These results are shown in Table 2
. Reactive sequences in gG-1, in comparison to the amino acid sequence of gG-2, are outlined in Fig. 2
, and the immunodominant regions for both proteins are shown, as well as the epitopes for the anti-gG-1 and anti-gG-2 MAbs. The intensity of the spots differed between the samples tested, suggesting the presence of varying amounts of antibodies directed against the mapped epitopes and/or different antibodypeptide affinities. Since the background reactivity of serum was too high, no conclusions could be drawn from these experiments and, consequently, only purified anti-gG-1 antibodies were employed. No cross-reactivity was seen when 10 of the purified human anti-gG-1 antibodies and the anti-gG-1 MAb were incubated with gG-2 peptides coupled to a cellulose membrane. As described previously (Liljeqvist et al., 1998
), purified human anti-gG-2 antibodies were non-reactive to gG-1 peptides. Neither HSV-negative sera (six sera tested) nor one HSV-2-isolation-proven serum, purified on the gG-1 antigen-coupled column, showed any reactivity to the gG-1 pepscan membrane (data not shown).
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Discussion |
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The reason for the retained type-specificity in regions with high similarity might depend on the existence of type-specific amino acids with key functions in antibody binding. As an example, some linear peptides derived from homologous parts of gC were recently shown to be presented in a type-specific manner (Ackermann et al., 1998 ). The importance of key residues has previously been shown in the work of Dolter et al. (1992)
for mouse MAb reactivity to HSV gC-1. Here, a mutation of only one amino acid in a MAb-binding region could change the specificity from HSV-1 to HSV-2. It is notable that the region in gG-2 that corresponds to the immunodominant region in gG-1 contains no epitopes for human antibodies, only epitopes for mouse MAbs (Liljeqvist et al., 1998
). On the other hand, nine of eleven purified human gG-1 sera tested reacted to the part of gG-1 that corresponds to the immunodominant region of gG-2, but without any sign of cross-reactivity to gG-2. This might be due to different spatial presentation and folding of these regions in gG-1 and gG-2, which in turn could be influenced by the pronounced difference in the degree of glycosylation of the two proteins. One might also speculate that the less extensive glycosylation of gG-1 argues for gG-1 being a well-exposed protein, easily accessible to the immune response, explaining why the epitopes in gG-1 are more dispersed compared with the restricted localization of human epitopes in gG-2 (Grabowska et al., 1999
; Liljeqvist et al., 1998
; Marsden et al., 1998
).
The pepscan method that was chosen has been used widely for the definition of antigenic determinants (Frank, 1992 ; Gao & Esnouf, 1996
; Kramer et al., 1994
; Reineke et al., 1996
). The most obvious drawback with the pepscan method is that it is selective towards reactivity to linear stretches of amino acids (Laver et al., 1990
), while epitopes are mostly discontinuous and of a complex, multi-dimensional structure (Van Regenmortel, 1995
, 1996
). Thus, structural alterations may occur when the polypeptide chain is produced as short peptide sequences anchored to a cellulose membrane. In this study, we have probably defined not only linear epitopes but also incomplete epitopes or linear parts of discontinuous epitopes. Another difficulty is setting the amino- and carboxy-terminal limitations of the epitopes, since the intensity of the spots on the membrane is dependent upon the concentration of the purified antibodies. Only purified anti-gG-1 antibodies were used in our experiments, since the background reactivity with sera was too high. Because of this, we can not exclude that additional epitopes would be found by using sera, as described by Liljeqvist et al. (1998)
for gG-2. When purifying sera, one also inevitably applies selection and suffers a loss of anti-gG-1 antibodies due to the purification and elution process. Another possible limitation is the selection of antibodies that occurs when using the recombinant, truncated gG-1 antigen for purification purposes. However, it is noteworthy that the strict HSV-1 type-specificity of the antibodies was retained and that the epitopes characterized can be ascribed to anti-gG-1 antibody activity, since both negative sera and an HSV-2 serum, purified on the gG-1 column, were negative in the pepscan analysis.
As regards HSV-1 type-specific serology, one disadvantage shown in earlier studies (Ashley et al., 1998 ; Lee et al., 1986
) is the relatively low sensitivity of immunoassays based on gG-1 antigen. Our ability to extract most of the anti-gG-1-reactive antibodies from the sera suggests that the small amount of these antibodies present in human sera might contribute to this phenomenon. In comparison, a total extraction of anti-gG-2 antibodies from patient sera is hard to achieve during purification and the total amount of anti-gG-2 antibodies in HSV-2 seropositive patients thus seems to exceed that of anti-gG-1 antibodies in HSV-1-seropositive patients. The relatively weak gG-1 band obtained in Western blots compared with the strong gG-2 signal also supports this. In our study, two sera showing low titres in the type-common ELISA were non-reactive when the purified anti-gG-1 antibodies were analysed in the type-common assay. The virus strain used as the antigen for the type-common ELISA is an HSV-1 laboratory strain that obviously does not express a large amount of gG-1. However, the purified anti-gG-1 antibodies were reactive in the gG-1 ELISA, showing that concentration of the antigen yielded a positive signal. Another prerequisite for an HSV-1 type-specific seroassay that has yet to be fulfilled is that the antigen should be based on a well-conserved gene among clinical HSV-1 isolates (Rekabdar et al., 1999
).
In conclusion, the human antibody reactivity to linear peptides representing HSV-1 gG-1 was predominantly localized to regions homologous to HSV-2 gG-2. In spite of this, the type-specific character of the purified anti-gG-1 antibody response was retained, indicating a type-unique presentation of the interspersed homologous amino acids to the immune system. Furthermore, the finding of an immunodominant region on gG-1 reactive to all sera tested and the non-reactivity of human anti-gG-1 antibodies to the corresponding stretches of amino acids on gG-2 suggests that this domain may be of interest in the further development of HSV-1 type-specific serology (Leinikki et al., 1993 ).
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Acknowledgments |
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References |
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Received 10 June 1999;
accepted 10 December 1999.