Department of Virology, Göteborg University, Guldhedsgatan 10 B, S-413 46 Göteborg, Sweden1
UPR9021 du CNRS, Institut de Biologie Moléculaire et Cellulaire, Strasbourg, France2
Author for correspondence: Jan-ke Liljeqvist. Fax +46 31 3424960. e-mail jan-ake.liljeqvist{at}microbio.gu.se
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Abstract |
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Introduction |
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In contrast to the well-described immunogenic properties of mgG-2, no information is available on the ability of sgG-2 to induce an antibody response in HSV-2-infected patients. We have previously presented evidence that the first 22 N-terminally located amino acids of the sgG-2 protein are a signal sequence and are cleaved off (Liljeqvist et al., 1999 ). The cleavage site of precursor gG-2 has not yet been determined, although it has been proposed that the molecule is cleaved between the amino acids arginine321 and alanine322 as well as between arginine342 and leucine343, where both sites are necessary for correct cleavage (R. Courtney, personal communication). The cleavage and processing pathway has been shown to be independent of other HSV-2 gene products (Su & Courtney, 1988
) suggesting that the cleavage events were mediated by a host cell-specific protease.
As sgG-2 contains multiple positively charged residues (McGeoch et al., 1987 ), the protein was purified from virus-infected cell medium by using a heparin column. The sgG-2 protein was used for production of monoclonal antibodies (mAbs), which were epitope mapped using a Pepscan technique based on peptides coupled to a cellulose membrane support (Frank, 1992
; Kramer et al., 1994
). The anti-sgG-2 mAbs presented no cross-reactivity to HSV-1-infected cells. Furthermore, since sgG-2 was successfully used as an antigen in ELISA for detection of type-specific antibodies from HSV-2-infected patient sera this protein may be suitable as a novel serological antigen for type-discriminating serology.
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Methods |
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Purification of sgG-2.
GMK-AH1 cells were infected with HSV-2 and when complete cytopathic effect was seen the medium was centrifuged at 2000 g for 10 min followed by ultracentrifugation at 100000 g for 1·5 h. The supernatant was concentrated by using microconcentrator tubes (Filtron Skandinavia) before the sample was applied to a HiTrap heparin column (Amersham Pharmacia) and recirculated for 2 h. After washing, a stepwise elution with increasing concentrations of NaCl (0·22·0 M) was performed and the fractions were concentrated with microconcentrator tubes until dryness. The proteins were resuspended in 200 µl PBS.
Production of mAbs.
Heparin-purified sgG-2 (100 µg) was emulsified in Freunds complete adjuvant for priming and in incomplete adjuvant for booster doses and injected intramuscularly to five female BALB/c mice at 3 week intervals. A sixth mouse was given a third immunization with immunoaffinity chromatography-purified sgG-2 protein. The fusion procedure followed standard hybridoma techniques (Fazekas de St Groth & Scheidegger, 1980 ) using the myeloma Sp2/O cells as fusion partner. The supernatants of the hybridomas were screened for antibody reactivity by ELISA and positive hybridomas were cloned by limiting dilution. The mAbs were cultured in dialysis tubing for large-scale production (Sjögren-Jansson & Jeansson, 1985
). Subclass specificity was determined by radial immunodiffusion (The Binding Site Ltd).
Immunoaffinity chromatography purified sgG-2.
The anti-sgG-2 mAb 4.G2.G10 (5 mg) was coupled to 2 ml of cyanogen bromide-activated Sepharose 4B (Amersham Pharmacia) according to the manufacturers instructions. Medium from virus-infected GMK-AH1 cells was harvested as described above and 0·3 M NaCl and 0·5% Nonidet P-40 were added before application to the column. The sample was recirculated for 1 h followed by washing with Tris-buffered saline (TBS) containing 0·5 M NaCl. The proteins were eluted with 0·1 M glycineHCl buffer (pH 2·8) and neutralized with TrisHCl (pH 8·0). The protein concentration was measured by DCProteinAssay (Bio-Rad).
Reactivity of anti-sgG-2 mAbs in an ELISA.
Immunoaffinity-purified sgG-2 (2·8 mg/ml) was coated at a 1:10000 dilution in carbonate buffer (pH 9·6) at 4 °C overnight on Maxisorp microtitre plates (Nalge Nunc). The plates were blocked with 2% skim milk in PBS for 1 h at 37 °C. The antibodies, at an initial concentration of 20 µg/ml, were diluted in twofold steps in PBS containing 1% skim milk and 0·05% Tween 20. After washing, peroxidase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories) was added at a 1:1000 dilution followed by addition of O-phenylenediamine as the substrate. In addition, the anti-sgG-2 mAbs and the anti-mgG-2 mAb O1.C5.B2 were tested against Helix pomatia lectin-purified mgG-2 as described previously (Liljeqvist et al., 1998 ). Supernatant from GMK-AH1 cells infected with HSV-1 was clarified and concentrated as described above, coated at a 1:100 dilution on microtitre plates, and the reactivity of the anti-gG-2 mAbs was tested in parallel. The results were given as end-point titres that were expressed as the reciprocal of the dilution giving an absorbance value greater than cut-off. Cut-off was defined as the reactivity to an unrelated antigen (cytomegalovirus) plus three SD.
Immunoblot.
Immunoaffinity-purified sgG-2 (8 µg) was diluted in 2% SDS including mercaptoethanol, boiled for 5 min, subjected to PAGE using 412% NuPAGE gradient gels (Novex), and electrotransferred to Immobilon-P transfer membrane (Millipore). Strips were incubated with anti-sgG-2 mAbs at a final concentration of 10 µg/ml. Peroxidase-labelled rabbit anti-mouse IgG (Dako) at a 1:100 dilution was used as conjugate with 4-chloro-1-naphthol as the substrate. For one of the mAbs, the reactivity to antigen subjected to SDSPAGE under non-reducing conditions was tested. In addition, cell lysates of HSV-2-infected GMK-AH1 and HEp-2 cells were subjected to SDSPAGE under reducing conditions as described earlier (Liljeqvist et al., 1999 ). The anti-mgG-2 mAb O1.C5.B2 and the anti-sgG-2 mAb 4.A5.A9 were tested for reactivity to the mgG-2 and the sgG-2 proteins. In addition, sera from ten HSV-2 isolation proven patients (see below) were tested for reactivity to the sgG-2 protein.
Indirect immunofluorescence.
Monolayers of GMK-AH1 and HEp-2 cells were cultivated on Lab-Tek chamber slides (Nunc Nalge) and infected with HSV-1 or HSV-2. When 25% of the cells were infected, the cultures were permeabilized and fixed in cold acetone for 5 min, washed in distilled water, dried and kept at -70 °C until use. For detection of membrane fluorescence, the cells were fixed with cold methanol for 10 min after addition of the mAbs. The anti-gG-2 mAbs were diluted in PBS (1 µg/ml) and incubated for 1 h at 37 °C. Fluorescein isothiocyanate-labelled F(ab')2 goat anti-mouse IgG (Jackson ImmunoResearch Laboratories) was used as conjugate at a 1:40 dilution and incubated for 1 h at 37 °C. The slides were mounted with a glycerolwater solution (4:1), and examined under a Nikon fluorescence microscope.
Nucleotide sequencing of the gG-2 gene.
As the HSV-2 strain B4327UR was used for the production of sgG-2, the segment of the gG-2 gene encoding the 342 amino-terminally located amino acids was sequenced following PCR amplification using methods and primers described elsewhere (Liljeqvist et al., 1999 ). The nucleotides were compared with the HSV-2 reference strain HG52 (McGeoch et al., 1987
).
Pepscan analysis of anti-sgG-2 mAbs.
The anti-sgG-2 mAbs were screened for binding using a panel of overlapping peptides (13-mers with 10 amino acid overlaps) spanning amino acids 23 to 342 (Jerini Bio Tools). Selected 15-mer peptides, overlapping by 14 amino acids, were synthesized for a more precise localization of the epitopes. The cysteine residues were replaced by serines to avoid oxidation during the incubation steps. The peptide sequences were deduced from nucleotide sequence data derived from strain B4327UR. The membranes were washed once in methanol and three times in TBS containing 0·05% Tween 20, pH 8·0 (T-TBS) and incubated overnight with blocking buffer (Genosys Biotechnologies) diluted 1:10 in T-TBS with addition of 50 mg/ml of sucrose. The mAbs were diluted (0·1 µg/ml) in blocking buffer and incubated for 3 h at room temperature. After triple washing, peroxidase-conjugated rabbit anti-mouse IgG (0·2 µg/ml), (Dako) was added in blocking buffer and incubated for 2 h. After washing, the membranes were developed with ECL detection solution on X-Omat S film.
For mapping of the anti-sgG-2 mAb 4.G2.G10, horseradish peroxidase was directly conjugated to the antibody using EZ-Link Maleimide activated horseradish peroxidase (Pierce). After purification, the mAb was incubated, at a concentration of 5 µg/ml, with the Pepscan membrane overnight at 4 °C. After triple washing for 5 min the ECL detection solution was added for development.
Competitive indirect ELISA.
15-mer peptides, comprising the predicted epitopes, were synthesized using 9-fluorenylmethoxycarbonyl chemistry (kindly provided by M. Levi, Karolinska Institutet, Stockholm, Sweden). The peptides were incubated at different concentrations with the corresponding mAbs for 18 h at room temperature. The mAb concentrations were selected from the end-point titration curves and close to the inflection points. The mixtures were assayed as described above in an indirect ELISA with sgG-2 as target antigen. The apparent dissociation constant value (KD) was calculated from the plot and expressed as the concentration of the peptide reducing the reactivity of the respective mAb to sgG-2 by 50% (Friguet et al., 1985 ) with correction for the bivalence of the mAbs (Stevens, 1987
). For the mAb 4.G2.G10, which recognized a non-linear epitope, three peptides including the mapped reactive amino acid stretches were mixed and tested as noted above.
Type-specific serology.
Immunoaffinity-purified sgG-2 protein was coated on Maxisorp microtitre plates as described above. Fifty sera from patients with an isolation-proven HSV-2 infection were tested. These sera were characterized by Western blot and identified both the mgG-2 protein and the carboxy-terminal high-mannose intermediate portion of gG-2. In addition, 25 sera from isolation-proven HSV-1-positive patients were included for analysis. These sera presented reactivity to a type-common sodium deoxycholate-solubilized membrane preparation of HSV-1 in an ELISA (Jeansson et al., 1983 ; Liljeqvist et al., 1998
; Svennerholm et al., 1984
), but were unreactive to Helix pomatia lectin-purified mgG-2. Finally, 25 HSV-negative patient sera were selected based on lack of reactivity to both the type-common HSV-1 antigen and to Helix pomatia lectin-purified mgG-2. Sera were tested in duplicate at a 1:100 dilution in PBS containing 0·6 M NaCl, 1% skim milk and 0·05% Tween 20, and incubated overnight at 4 °C. Peroxidase-conjugated goat anti-human IgG (Jackson ImmunoResearch Laboratories) was added at a 1:3000 dilution, and O-phenylenediamine was used as the substrate. The reaction was stopped with 1 M sulfuric acid and the absorbance was measured at 492 nm.
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Results |
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For the production of anti-sgG-2 mAbs, five fusions were performed after using the heparin-purified sgG-2 eluted with 0·5 M NaCl as immunization antigen. With this protocol, the mAbs 4.G2.G10 and 4.A5.A9 were detected. Immunoaffinity-purified sgG-2 (mAb 4.G2.G10) was used for booster immunization of the sixth mouse. In the subsequent fusion, the mAbs 8.A10.F10, 5.C3.C6 and 6.D12.B3 were produced. The mAbs were of IgG1 subclass.
Identification and localization of sgG-2 and mgG-2
Four anti-sgG-2 mAbs were reactive in immunoblot when sgG-2 was subjected to SDSPAGE under reducing conditions (Fig. 1a). MAb 4.G2.G10 showed reactivity to sgG-2 only when SDSPAGE was performed under non-reducing conditions. This finding suggests that the mAb recognized a non-linear epitope of which at least one disulfide bond is essential to maintain the three-dimensional integrity of the epitope.
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The cellular localization of the gG-2 proteins was also tested by indirect immunofluorescence with HSV-2-infected GMK-AH1 and HEp-2 cell membranes or permeabilized cells. The reactivity was essentially identical for the two cell-lines. The anti-mgG-2 mAb recognized the mgG-2 protein both on virus-infected cell membranes and in the cytoplasm of permeabilized cells (Table 1). All the anti-sgG-2 mAbs clearly identified the sgG-2 protein, and as judged from the immunoblot experiment to a minor extent the precursor protein (see Fig. 1b
), in the cytoplasm of permeabilized HSV-2-infected cells while no reactivity was seen to virus-infected cell membranes.
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Type-specific reactivity of anti-gG-2 mAbs
The anti-sgG-2 mAbs were all reactive to sgG-2, presenting variable end-point titres in an ELISA (Table 1). The anti-sgG-2 mAbs were unreactive to mgG-2 and to culture medium from HSV-1-infected cells. By indirect immunofluorescence, all anti-gG-2 mAbs were unreactive to HSV-1-infected cell membranes as well as to permeabilized cells, indicating that the mAbs recognized HSV-2 type-specific epitopes.
Identification of linear epitopes of sgG-2
The synthetic peptides used in the Pepscan analysis were deduced from nucleotide sequence data of strain B4327UR. The following nucleotide differences were found in strain B4327UR as compared with strain HG52 (McGeoch et al., 1987 ) (numbering refers to strain HG52): G
A104, altering amino acid serine35 to asparagine, T
C274 (silent mutation), G
A329, giving replacement of arginine110 by histidine, a deletion of GTC879 encoding valine293, and finally C
T930 (silent mutation). Consequently, only two amino acid alterations were included in the synthesized peptides as compared to strain HG52.
For screening purposes, the mAbs were mixed and tested for reactivity to a cellulose membrane with 13-mer peptides with 10 amino acid overlaps spanning amino acids 23 to 342. Two stretches of the sgG-2 protein, localized within the carboxy-terminal part of the protein, were shown to exhibit antibody reactivity (Fig. 2a). The respective mAbs were fine-mapped by using separate membranes covering the reactive regions (15-mer peptides with 14 amino acid overlaps). Four antibodies presented distinct reactivities to the peptides whereby the respective epitopes were determined. The length of the epitopes varied between three to five residues and the sequences and their localization in sgG-2 are illustrated in Fig. 2(b)
.
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Reactivity of human sera to sgG-2
Immunoaffinity-purified sgG-2 was evaluated as an antigen in an indirect ELISA under specific conditions described in Methods. Fifty sera from isolation proven HSV-2-infected patients presented absorbance values in the range 0·21 to1·86 (Fig. 4). Sera from 25 isolation proven HSV-1-infected patients and sera from 25 HSV-negative patients presented significantly lower values in the range 0·01 to 0·07.
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Discussion |
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A major HSV-1/HSV-2 difference in gene length is found between the two gG genes where HSV-1 lacks a stretch homologous to sgG-2. The sgG-2 protein has therefore the potential to elicit an exclusively type-specific B-cell immune response in the host. Here we showed that the anti-sgG-2 mAbs presented a type-specific reactivity to HSV-2 with no cross-reactivity to HSV-1 antigen, and that the sgG-2 protein was immunogenic and evoked a type-specific antibody response in HSV-2-infected patients. This protein may therefore be suitable as an additional serological antigen for detection of anti-sgG-2 antibodies. As sgG-2 is secreted rapidly during replication, the antibody response to sgG-2 may be elicited earlier after infection. However, until the performance of sgG-2 is investigated in large serological studies including early infections, the mgG-2 protein should be considered as the prototype antigen for type-specific serology.
In the mapping of HSV epitopes, antibody reactivity to antigen subjected to SDSPAGE and immunoblot has been used for discrimination of linear from non-linear epitopes. In the latter case denaturing and reducing agents disrupt the integrity of the epitope and usually no antibody reactivity is detected under these conditions (Muggeridge et al., 1990 ). The distinction may be valuable for selection of mapping strategy as antibodies recognizing linear epitopes can usually be localized by synthetic peptides in contrast to non-linear or discontinuous epitopes which are unreactive (Isola et al., 1989
; Jemmerson, 1987
). However, a few studies have recently detected non-linear epitopes by the use of Pepscan techniques based on peptides coupled to Immobilon (Gao & Esnouf, 1996
) or to cellulose membranes (Korth et al., 1997
; Reineke et al., 1995
, 1998
).
The non-linear epitope described for mAb 4.G2.G10 was composed of three discrete peptide stretches of sgG-2. In a previous study, Su et al. (1993) produced a rabbit hyperimmune serum against a peptide derived from the sgG-2 sequence. This peptide sequence overlapped with one of the reactive peptide stretches described here for the non-linear epitope (245GLRFRER251). These observations indicate that this region of sgG-2 is exposed and immunogenic in both mouse and rabbit. The epitope contained mostly positively charged arginines and hydrophobic residues compatible with data obtained from X-ray crystallography analysis of antibodyantigen interactions showing that non-linear epitopes usually are composed of highly charged amino acids flanking a central core of hydrophobic residues (Kwong et al., 1998
; Padlan et al., 1989
; Smith et al., 1996
). The most carboxy-terminal reactive peptide stretch of the epitope described for mAb 4.G2.G10 was amino acids 320RRAL323. This motif overlapped with the amino-terminally localized cleavage site which is proposed to occur between arginine321 and alanine322. One explanation for this finding might be that small amounts of the gG-2 precursor protein are secreted into the extracellular medium or that the cleavage site is not utilized in the cell line used here.
Several mammalian alphaherpesviruses sequenced to date code for positional gG homologues which are secreted (Crabb et al., 1992 ; Keil et al., 1996
; Rea et al., 1985
; Telford et al., 1992
). The gG-2 gene has been shown to be non-essential for replication of HSV-2 in vitro (Harland & Brown, 1988
) and in vivo for isolates from the recurrent HSV-2 lesions of some rare patients (Liljeqvist et al., 1999
). Furthermore, mutants of pseudorabies virus lacking the gene encoding the secreted gG homologue (gX) were shown to be fully virulent in pigs with induction of a complete protective immunity (Kimman et al., 1992
). Thus, the functions of these gene products have hitherto been elusive. An interesting observation is that sgG-2 was not only secreted into cell-culture medium but also accumulated in lysates of HSV-2-infected cells, a finding which suggests that the protein may also have an intracellular function within HSV-2-infected cells. In addition to the potential use of sgG-2 as a novel HSV-2-specific serological antigen, the production and mapping of anti-sgG-2 mAbs described in this study may be a valuable tool facilitating future studies addressing possible intra- and extracellular interactions of sgG-2.
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Acknowledgments |
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Footnotes |
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Received 29 June 2001;
accepted 17 September 2001.