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Journal of Clinical Microbiology, August 1999, p. 2717-2718, Vol. 37, No. 8
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Typing of Clinical Herpes Simplex Virus Type 1 and
Type 2 Isolates with Monoclonal Antibodies
Jan-Åke
Liljeqvist,*
Bo
Svennerholm, and
Tomas
Bergström
Department of Virology, University of
Göteborg, S-413 46 Göteborg, Sweden
Received 17 December 1998/Returned for modification 7 February
1999/Accepted 21 April 1999
 |
ABSTRACT |
The purpose of this study was to evaluate the performance of a
herpes simplex virus (HSV) type 1-specific anti-glycoprotein C-1
monoclonal antibody (MAb) and a type 2-specific anti-glycoprotein G-2
MAb for typing of 2,400 clinical HSV-1 isolates and 2,400 clinical
HSV-2 isolates, respectively, using an enzyme immunoassay. The
anti-HSV-1 MAb showed sensitivity and specificity of 100%, and the
anti-HSV-2 MAb showed a sensitivity of 99.46% and 100% specificity,
indicating that these MAbs are suitable for typing of clinical HSV isolates.
| |
TEXT |
Herpes simplex virus (HSV) types 1 and 2 are ubiquitous human viruses for which a correct subtyping is
essential for counseling work, epidemiological studies, and follow-up
programs in vaccine trials. Virus culture, followed by antigenic
detection of type-specific determinants, is often used as the "gold
standard" to discriminate HSV-1 from HSV-2 (1, 16). Since
several HSV-specific monoclonal antibodies (MAbs) have been shown to
identify type-specific epitopes (2, 5, 11, 17), these
reagents have been used for type-specific antigen detection. However,
the reported sensitivities and specificities for such reagents,
including commercially available kits, have been found to vary among
the assays in the range between 92 and 100% (4, 6, 7, 17).
This discrepancy may be explained by variability in the expression of
different HSV proteins or intratypic variability of different epitopes
within the same protein among the isolates (11). Therefore,
the selection of the type-specific MAbs may be an important determinant
of the sensitivity and specificity of the assay. Another explanation
could be that only a restricted number of isolates (in the range
between 38 to 119) have been tested in the above studies
a factor
which, statistically, may significantly influence the accuracy of the test.
The purpose of this study was to investigate the sensitivity and
specificity for an HSV-1-specific anti-glycoprotein C-1 (gC-1) MAb
(B1.C1) and an HSV-2-specific anti-glycoprotein G-2 (gG-2) MAb
(O1.C5.B2) in the typing of 2,400 clinical HSV-1 isolates and 2,400 clinical HSV-2 isolates, respectively. The MAbs used were produced at
our laboratory as described earlier (5, 9), and in contrast
to other type-specific anti-HSV MAbs used for typing for which data
have been published, these have been mapped in detail. The anti HSV-1
MAb B1.C1 has been shown to bind to antigenic site II in gC-1, in which
threonine150 is crucial for binding (10). In
addition, the MAb interferes with the heparan sulfate-binding activity
of gC-1 (15), thereby inhibiting attachment of the virus to
cells (14). The anti-gG-2 MAb epitope was recently mapped to
the residues HRGGPEE, a stretch of amino acids which is localized
within an immunodominant region for which all tested HSV-2-positive
human sera were reactive (5).
HSV isolates from patients with clinical lesions, consecutively
received at the Department of Clinical Virology in Göteborg, Sweden, were cultured on GMK-AH1 cells. Positive cultures were subtyped
on GMK-AH1-infected cells by an enzyme immunoassay until a total number
of 2,400 HSV-1 and HSV-2 isolates each was reached. Confluent
monolayers of GMK-AH1 cells were infected with each isolate in 96-well
plates. When complete cytopathic effect was seen, the cells were fixed
in 0.25% glutaraldehyde in phosphate-buffered saline for 30 min. The
type-specific MAbs and an HSV type-common anti-gE MAb (B1.E6) (2,
8) were added at a 1:100 dilution (initial concentration of 100 µg/ml) and incubated for 1 h at room temperature. Alkaline
phosphatase-conjugated F(ab')2 goat anti-mouse
immunoglobulin (Jackson Immuno Research Labs) at a 1:2,000 dilution was
used as conjugate, with p-nitrophenyl dissolved in carbonate
buffer (pH 9.8) as a substrate. The reaction was read at 405 nm after
30 min. Clinical isolates with reactivity with neither of the two
type-specific MAbs were typed by PCR technique by using primers from
the type-unique promoter region of the glycoprotein D gene
(13). Clinical HSV isolates were defined as unreactive for
any of the MAbs if the absorbance values (optical density [OD] units)
were less than 0.2 OD units above the reactivity of uninfected cells.
The local HSV-2 strain B4327UR (S. Jeansson, Göteborg, Sweden)
and the HSV-1 strain KOS321 were used as positive control viruses.
Of the 4,800 tested HSV isolates (2,400 of each subtype), 13 showed no
reactivity with the two type-specific MAbs but a clear reactivity with
the type-common anti-gE MAb. These isolates were confirmed as HSV-2
positive with PCR. All isolates but three were reactive to the anti-gE
MAb; one isolate was identified as HSV-1 and two isolates were
identified as HSV-2 based on the reactivities of the type-specific MAbs
and PCR findings. In total, four isolates were reactive to all three
MAbs used and were confirmed to be both HSV-1 and HSV-2 by PCR and
therefore judged to reflect dual infections. These findings are in
agreement with those of other studies in which dual infections for the
same lesion were found to be rare events (3, 7).
The anti-gC-1 MAb recognized all HSV-1 isolates, giving a sensitivity
of 100%. There was a highly statistically significant difference
between gC-1 and gG-2 used as target proteins (P < 0.001, chi-square test), indicating that the intratypic
variability within these two epitopes differed among the clinical HSV
isolates. Since the gC-1 protein was described earlier to be
efficiently expressed at the cell surface (12), this protein
has been suggested as a suitable target antigen for diagnostic MAbs
(17). The domain recognized by the anti-gC-1 MAb was here
shown to be an ideal target for the typing of HSV-1 isolates since the
epitope was found to be highly conserved among different clinical HSV-1
isolates. This conservation may reflect the essential function of
heparan sulfate binding for the virus in vivo. Despite the fact that
the MAb binds to a region with significant homologies to gG-2, no cross-reactivity to HSV-2 isolates was seen, indicating that gC-1 and
gG-2 are structurally different within the epitope detected by the
B1.C1 MAb. Although type-specific anti-gC-1 MAbs have previously been
shown not to cross-react with HSV-2 isolates (7, 17), other
workers (11) have described such a cross-reactivity,
suggesting an intratypic variability for different epitopes within
gC-1. A problem discussed earlier in a study comparing commercial kits used for typing of HSV isolates was that MAbs specific for HSV-2 were
somewhat cross-reactive with HSV-1 isolates (7). One
advantage of using gG-2 as the target protein for the typing of HSV-2
isolates could be that only type-specific epitopes have been described in gG-2 and that human anti-gG-2 antibodies and anti-gG-2 MAbs do not
cross-react to native or denatured HSV-1 antigen as well as to
homologous gG-1 peptides (5). The earlier reported HSV-2 type-specificity of the anti-gG-2 MAb (O1.C5.B2) was confirmed in this
study since this antibody was unreactive to all tested clinical HSV-1
isolates. Only 13 (0.54%) of 2,400 HSV-2 isolates were unreactive to
the anti-gG-2 MAb. Therefore, this epitope also seems highly conserved
among clinical HSV-2 isolates.
In summary, this study describes the good performance, with both high
sensitivity and high specificity, of an anti-gC-1 MAb and an anti-gG-2
MAb in the subtyping of a great number of clinical HSV isolates. These
well-characterized MAbs may also be tested as reagents for rapid
detection of type-specific HSV antigen directly in clinical specimens.
| |
ACKNOWLEDGMENTS |
This work was supported by grants from the Medical Society of
Göteborg, Swedish Medical Research Council (MFR, grant 11225), the LUA Foundation at Sahlgren's Hospital, the Central Committee for
Animal Research (CFN, centrala försöksdjursnämnden),
and the Swedish Society for Medical Research.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Virology, University of Göteborg, Guldhedsgatan 10 B, S-413 46 Göteborg, Sweden. Phone: 46 31 3424657. Fax: 46 31 3424960. E-mail: jan-ake.liljeqvist{at}mednet.gu.se.
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Journal of Clinical Microbiology, August 1999, p. 2717-2718, Vol. 37, No. 8
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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