Type-specific reactivity of anti-glycoprotein G antibodies from herpes simplex virus-infected individuals is maintained by single or dual type-specific residues

Petra Tunbäck1,2, Tomas Bergström2, Gun-Britt Löwhagen1, Johan Hoebeke3 and Jan-Åke Liljeqvist2

1 Department of Dermatovenereology, Göteborg University, Guldhedsgatan 10B, 413 46 Göteborg, Sweden
2 Department of Clinical Virology, Göteborg University, Guldhedsgatan 10B, 413 46 Göteborg, Sweden
3 UPR9021 du CNRS, Institut de Biologie Moléculaire et Cellulaire, Strasbourg, France

Correspondence
Jan-Åke Liljeqvist
jan-ake.liljeqvist{at}microbio.gu.se


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Glycoprotein G-1 (gG-1) of herpes simplex virus type 1 (HSV-1) and gG-2 of HSV-2 are the only known HSV proteins that induce type-specific human antibody responses. Recently, it was shown that purified human anti-gG-1 and anti-gG-2 antibodies presented a type-specific reactivity to immunogenic stretches with high similarity between gG-1 and gG-2. In this study, the molecular basis for this type-specific recognition was investigated employing synthetic peptides covering the indicated regions, including substitutions of the type-specific residues. The results revealed that single or dual type-specific residues localized within regions of high similarity could induce significant structural differences, explaining the type-specific recognition of the human antibody response to the gG proteins.


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Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) are closely related human viruses belonging to the family Herpesviridae. Eleven HSV envelope glycoproteins have been defined and several of these are strong inducers of the human antibody response. Although the amino acid similarity between HSV-1 and HSV-2 is high, the envelope glycoprotein G (gG) proteins are the only HSV antigens known to induce type-specific antibody responses and are therefore used in serological assays to discriminate between HSV-1 and HSV-2 infection (Ashley et al., 1991; Ashley, 1998; Görander et al., 2003; Sánchez-Martínez et al., 1991). The reason for this is that the genes encoding the gG proteins have evolved differently and gG-1 of HSV-1 contains 238 aa, while gG-2 of HSV-2 comprises 699 aa (McGeoch et al., 1985, 1987) (Fig. 1). Furthermore, the gG-2 protein is unique among the HSV proteins in that it is cleaved into one secreted part (sgG-2) and one portion anchored to the virion (mgG-2).



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Fig. 1. Schematic presentation of the gG-1 and gG-2 proteins. Dark grey bars represent regions with an overall sequence similarity of approximately 50 %. The localization of the mapped human epitopes is shown by white boxes. The amino acid sequences for the immunogenic regions of gG-1 and mgG-2 investigated in this study are displayed and identical amino acids are depicted in bold. TMR, transmembranous region.

 
Earlier studies using the pepscan technique demonstrated that immunosorbent-purified human anti-gG-1 and anti-mgG-2 antibody samples recognize linear stretches of these proteins (Liljeqvist et al., 1998; Tunbäck et al., 2000). Within the mapped gG-1 and mgG-2 human epitopes, only one stretch with high similarity was outlined, carrying nine type-common and five type-specific residues (Fig. 1). The corresponding stretches of amino acids were found to be immunogenic, eliciting an antibody response in almost all HSV-1- and HSV-2-infected individuals. In spite of this similarity, the type-specific reactivity of purified anti-gG-1 and anti-mgG-2 polyclonal antibodies was maintained (Tunbäck et al., 2000).

In this study, sera were collected from six isolation-proven patients with HSV-1 infection and six patients with HSV-2 infection. One patient was double infected and this sample was included in both groups. The HSV-1 isolates were collected from orolabial lesions and HSV-2 isolates from genital lesions. Serum samples were assayed in a type-specific HSV-1 ELISA using gG-1 as antigen and a type-specific HSV-2 ELISA using mgG-2 as antigen (Svennerholm et al., 1984; Tunbäck et al., 2000). Results showed that sera from isolation-positive HSV-1 patients were positive in the type-specific HSV-1 ELISA and sera from the HSV-2-positive patients were reactive in the HSV-2 ELISA. Results were verified by immunoblotting. A recombinant truncated gG-1 (kindly provided by Smith-Kline Beecham) and Helix pomatia lectin-purified mgG-2 (Olofsson et al., 1986) were coupled to epoxy-activated Sepharose (Pharmacia Biotech) and sera were added to the columns in order to achieve purified anti-gG-1 and anti-mgG-2 antibodies (Liljeqvist et al., 1998; Tunbäck et al., 2000). Purified anti-gG-1 samples were reactive in the HSV-1 ELISA, but non-reactive in the HSV-2 ELISA and vice versa. The study protocol was approved by the Ethics Committee of the Medical Faculty of Göteborg University.

The immunodominant region of mgG-2 has been mapped to P552–E574 (Liljeqvist et al., 1998). The homologous part of the gG-1 sequence (aa E80–L93) was also involved in binding for the majority of human-purified anti-gG-1 antibody samples (Tunbäck et al., 2000) (Fig. 1). Although these regions are highly similar, purified antibodies from an HSV double-infected patient presented no cross-reactivity. Here, we analysed peptides covering these regions, carrying amino acid substitutions from the corresponding gG sequences. The sequences of the peptides were based on earlier studies using DNA sequencing of the gG-1 and mgG-2 genes of clinical HSV isolates (Liljeqvist et al., 2000; Rekabdar et al., 2002). Purified antibodies were added to the gG-1 and mgG-2 peptides and analysed as described earlier (Liljeqvist et al., 1998). Binding was determined by visual assessment in which the intensity of the spots was scored (+/++/+++).

Purified anti-gG-1 antibodies from the HSV-1-infected patients (Table 1, patients 1–6) were added to peptides covering the mgG-2 region, containing amino acid substitutions from the gG-1 sequence (modified mgG-2 peptides). A distinct reactivity was seen for all samples to the control gG-1 sequence (peptide 14), but no cross-reactivity was present to the original mgG-2 peptide (peptide 1). It was evident that antibodies exhibited binding when peptides contained histidine and leucine from the gG-1 sequence (peptides 8 and 9) in an otherwise homotypic mgG-2 background. Thus, the type-specific reactivity of anti-gG-1 antibodies was dependent on the two key-residues H92 and L93. Consequently, the other gG-1 type-specific glutamate residues, E80, E81 and E85, were not involved in binding.


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Table 1. Reactivity of purified human anti-gG-1 and anti-mgG-2 antibodies against modified mgG-2 peptides and modified gG-1 peptides

Human antibody samples are numbered 1–11. Binding was scored from + to +++. Substituted amino acids are shown in bold.

 
When purified anti-gG-1 antibodies were added to the membrane carrying modified gG-1 peptides, all samples from HSV-1-infected patients (patients 1–6) showed retained binding as long as histidine and leucine (Table 1, peptides 1–5) were present. When either of these was exchanged for the corresponding mgG-2 residue proline, the capacity of antibodies to bind was abolished. However, one of the samples (patient 3) retained a weak binding, even when histidine was exchanged for proline from the mgG-2 sequence (H92->P, peptides 6, 11 and 12) as long as L93 was present. It was notable that the insertion of type-specific mgG-2 residues into the otherwise identical gG-1 peptide (peptides 2–5) did not diminish the anti-gG-1 antibody binding. These data suggested that the human antibody response to the gG-1 region was monospecific and that only the type-specific residues histidine and leucine were essential for binding.

Purified anti-mgG-2 antibodies from the HSV-2-positive samples (Table 1, patients 1 and 7–11) were added to modified gG-1 peptides. No cross-reactivity was detected to the original gG-1 sequence (peptide 1), while a clear reactivity was observed to the control mgG-2 peptide (peptide 14). Reactivity was present for all samples, although with variable intensity, when amino acids 92HL93 were both substituted by prolines (peptides 8 and 9) from the mgG-2 sequence, indicating their importance in binding. All samples exhibited a weak binding to modified gG-1 peptides when histidine was exchanged for proline (H92->P, peptides 6, 11 and 12). For two samples (patients 1 and 8), a weak reactivity was seen when E85 from gG-1 was substituted for phenylalanine (F85) from the mgG-2 sequence (peptide 2).

Purified anti-mgG-2 antibodies were added to membranes containing modified mgG-2 peptides. Four of the samples (patients 1, 7, 9 and 10) reacted to the modified mgG-2 sequence, as long as the C-terminal prolines of the peptide were preserved (Table 1, peptides 1–5). The other two samples (patients 8 and 11) were unable to bind when F565 was substituted for glutamate from the gG-1 sequence, even though the amino acids 572PP573 were present (peptide 2). Samples from all patients showed reactivity when the first of the two C-terminal prolines was preserved (peptides 7, 10 and 13). When the second of the two prolines was present, binding was detected to peptide 6 for samples derived from patients 1, 7 and 9, while no reactivity was seen to peptides 11 and 12, suggesting that the N-terminal 560GP561 residues were required for binding. One sample (patient 1) presented retained reactivity despite the fact that the C-terminal prolines were changed to 92HL93 from the gG-1 sequence, as long as the peptide contained the type-specific mgG-2 residues 560GP561 and F565 (peptide 8). We concluded that the human antibody response to the mgG-2 region seemed to be polyspecific and that all mgG-2 type-specific residues were involved in binding. Earlier studies, employing mAbs against HSV gC-1 and gC-2, showed that the type-specific reactivity was maintained by a single type-specific amino acid (Dolter et al., 1992; Kimmel et al., 1990). Here we have presented similar results for the type-specific behaviour of the human polyclonal antibody response to highly similar immunogenic regions of gG-1 and mgG-2.

To explore the possible conformers of the gG-1 and mgG-2 peptides in an aqueous environment, the INSIGHT II software (Accelrys) was used for molecular modelling on a Silicon Graphics workstation as proposed previously (Bartels et al., 1998). Peptides were minimalized for 5000 cycles by the conjugated gradient minimalization process and then subjected to a dynamic energy sampling at 1000 °K using 10 ps of equilibration and 100 ps of dynamics. Four conformers with minimal potential energy were selected and again minimalized by a conjugated gradient for 4000 steps (final derivatives less than 0·01 kcal–1 Å–1) showing totally different patterns of conformation (Fig. 2). The gG-1 peptide presented a restricted flexibility over the whole backbone, with the exception of histidine and leucine residues, which were shown to be essential for type-specific antibody recognition. The restricted flexibility is suggested to be due to the extended form induced by the poly-glutamyl N terminus, avoiding contact between the negatively charged residues and favouring contact of these residues with water molecules. In contrast, the mgG-2 peptide showed a high flexibility, which was made possible by the distribution of proline and glycine residues and lack of the poly-glutamyl N terminus present in gG-1.



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Fig. 2. Four superimposed molecular models of each of the investigated gG-1 and mgG-2 peptides in an aqueous environment. The amino acid sequences are shown vertically and the type-specific residues are displayed in bold.

 
Synthetic peptides are used to simulate antibody binding to native antigens. Due to the linear arrangement of the peptides, the pepscan technique is biased towards detection of linear epitopes (Laver et al., 1990). However, it is possible to recognize discontinuous epitopes demonstrated by reactivity to separated linear stretches of the protein. In that situation difficulties arise in inhibiting antibody reactivity to the native protein by adding relevant peptides in a competitive indirect ELISA (Liljeqvist et al., 2002). In studies of human polyclonal anti-gG-1 and anti-mgG-2 antibodies, inhibition of antibody reactivity to native antigens was shown at low concentrations of the peptides (Liljeqvist et al., 1998; Tunbäck et al., 2000). This suggests that the mapped epitopes are mostly linear and that the peptides used here mimic antibody reactivity to the native proteins. This conclusion may in part be explained by the fact that the extracellular portions of gG-1 and mgG-2 lack cysteine residues, which can be utilized for folding of proteins into complex conformations. It was notable that the mapping results described, using linear peptides, were obtained from antibodies recognizing the whole gG-1 and mgG-2 proteins. However, we cannot exclude the existence of anti-gG-1 and anti-mgG-2 antibodies recognizing discontinuous epitopes unable to be mapped with the pepscan method.

Molecular modelling results allowed interpretation of the experimental data regarding immunological characteristics of the gG-1 and mgG-2 peptides. Recognition and binding of antibodies are favoured by high mobility of the antigen (Westhof et al., 1984). In addition, homopolymers such as poly-L-glutamic acids are not immunogenic (Sela, 1966). The combination of these properties points towards the flexible histidine–leucine C terminus as the only target for antibody recognition and explains loss of binding if one of these amino acids is substituted. It can therefore be suggested that the anti-gG-1 antibody response to this region is monospecific. This is supported by data on reactivity of human anti-gG-1 antibodies, which showed that peptides containing the poly-glutamyl stretch but lacking the histidine and leucine residues were inert (Tunbäck et al., 2000). Histidine and leucine were therefore considered energetically essential, constituting a key function for binding. The mgG-2 peptide, in contrast, showed great flexibility in water, which opens up the possibility that several epitopic targets are present and explains the heterogeneous response between patients to different amino acid replacements (Table 1). With the knowledge that short peptides normally do not demonstrate secondary structures in water, no efforts were made to verify the predicted peptide conformations by nuclear magnetic resonance.

In conclusion, here we employed a substitution analysis of similar regions of gG-1 and mgG-2 using a pepscan technique to dissect the molecular basis for the type-specific reactivity of purified human anti-gG-1 and anti-mgG-2 antibodies. The type-specific polyclonal antibody response to the region of high similarity between gG-1 and mgG-2 investigated here is a prerequisite for using the proteins as antigens for serodiagnostic purposes in HSV-1 and HSV-2 infection. Results showed that the maintenance of type specificity was dependent on single or dual type-specific residues having a key function in binding. Molecular modelling data suggested that the experimental results could be explained by differences in flexibility and structure of the gG peptides, resulting in different exposure of key residues utilized for recognition.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from The Medical Society of Göteborg, The Swedish Society of Medicine, the LUA Foundation at Sahlgrenska University Hospital and The Swedish Research Council, grant no. 11225.


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Received 1 October 2004; accepted 3 November 2004.



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