JOURNAL OF VIROLOGY, Aug. 1994, p. 5284-5286 Vol. 68, No.

8
0022-538X/94/$04.00+0
Copyright © 1994, American Society for Microbiology

Cervical Antibodies in Patients with Oral Herpes Simplex Virus

Type 1 (HSV-1) Infection: Local Anamnestic Responses
after Genital HSV-2 Infection
RHODA ASHLEY,1* ANNA WALD,12 AND LAWRENCE COREY' 2'3
Departments of Laboratory Medicine, 1 Medicine,2 and Microbiology,3 University of
Washington School of Medicine, Seattle, Washington 98195
Received 19 November 1993/Accepted 22 April 1994

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Herpes simplex virus (HSV)-specific immunoglobulin A, immunoglobulin G, and secretory-component-
containing immunoglobulins were identified in cervical and salivary secretions from six subjects with oral HSV
type 1 (HSV-1) infections. Anamnestic cervical and salivary antibody responses were detected in two
HSV-1-seropositive women with newly acquired genital HSV-2 infections. These data implicate the common
mucosal immune system in antibody responses to HSV.

The common mucosal immune system provides local immu-
nity at the site of microbial infection and, in addition, is defined
by a redistribution scheme which populates distant mucosae
with stimulated lymphocytes (10, 13). If this system includes
the genital tract, antibodies to herpes simplex virus type 1
(HSV-1) should be present in genital secretions in response to
oral herpes. In turn, these local immune factors might play a
role in the partial resistance to the acquisition of HSV-2
genital infections by individuals with prior HSV-1 immunity
and in the limitation of the severity of these infections (4-6, 8,
12). A modified Western blot (immunoblot) technique was
used to analyze salivary and cervical secretions of eight HSV-
1-seropositive women for immunoglobulin A (alpha chain)
[IgA(ot)], IgG(y), and secretory-component-containing anti-
bodies (SC-Ig) which bind to HSV proteins.
This study was approved by the University of Washington
Human Subjects Review Committee. Subjects 1 through 6
were HSV-1-seropositive immunocompetent adults from
whom samples were taken weekly for 4 to 6 weeks. The
samples, which were taken from oral and genital mucosae,
were tested for both local antibodies and HSV. None of the six
subjects had genital herpes; three had oral recurrences, and
three remained asymptomatic. Subjects 7 and 8 were enrolled
at the University of Washington Virology Research Clinic, with
HSV-2 genital infections which were confirmed by culture (15)
and by subsequent seroconversion to HSV-2 (3). Both subjects
presented on their third day of illness (day 3) with genital
lesions, cervicitis, and neck stiffness (subject 7) or urethritis
(subject 8). Both began acyclovir (200 mg five times daily for 10
days) on day 3. Specimens for viral isolation were collected
from the cervix, vulva, urine, and rectum at enrollment; then at
2- to 4-day intervals until lesions healed at days 11 and 13; and
weekly, thereafter, for eight weeks. HSV-2 was isolated from
multiple sites from both subjects on day 3. On day 5, cervical
cultures from subject 7 and cervical and vulvar cultures from
subject 8 were positive for HSV-2. Cultures were negative
thereafter.
Cervical secretions were sampled at each visit by a previ-
ously described wicking technique. Expectorated saliva was
centrifuged for 15 min at 12,000 x g. IgA, IgG, and protein
concentrations in occult blood-negative secretions were deter-
mined (2a). Saliva was diluted 1:10 and cervical secretions
were diluted 1:40 before Western blotting with enhanced
chemiluminescence was performed for detection of HSV-
specific IgA, IgG, and IgM (7) and of SC-Ig with anti-human
secretory component (13a). Reactive bands were identified
and scored without knowledge of the subjects' HSV status.
Cervical IgA, IgG, and SC-Ig to oral HSV-1. The patients
with oral HSV-1 infections, subjects 1 through 6, all had
cervical IgG, IgA, and SC-Ig (Table 1). IgA and SC-Ig profiles
had fewer bands and were less intense than IgG profiles,
despite the finding that total cervical IgA concentrations were

TABLE 1. HSV-1 protein-specific IgA, IgG, and SC-Ig in cervical secretions of subjects with oral HSV-1 infections
Cervical IgA Cervical IgG
Subject Cervical SC-Ig, HSV-1
target(s)j
HSV-1 Total IgA
concnb
target(s) HSV-1 target(s) TotlI
~~~~~~~~~~~~~~~~~~~~concnb
1 gC 14.8 VP5, gC, gD VP5, gB, gC, gE, VP16, gD 8.6
2 VP5, gB, gC, gE, gD 17.0 VP5, gB, gC, gE, gD All 7 14.5
3 VP5, gB, gC, gD 10.8 gB, gC, gD, ICP35 All 7 3.4
4 gB, gC, gD 7.5 VP5, gB, gC, gD, ICP35 gB, gC, gE, VP16, gD, ICP35 1.2
5 gC, VP16, gD 13.3 gB, gC, gE, gD gB, gC, gE, VP16, gD, ICP35 8.4
6 VP5, gB, gC, gE 28.9 VP5, gD All 7 6.3
HSV-1 protein targets were VP5, gB, gC, gE, VP16, gD, and ICP35.
b
Expressed in micrograms per 100 ,lp.
*
Corresponding author. Mailing address: University of Washington,
Children's Hospital & Medical Center, CH82, 4800 Sand Point Way
N.E., Seattle, WA 98105.
5284
VOL. 68, 1994 NOTES 5285

cervical salivary salivary IgG concentration ranged from 0 to 0.3 ,ug/100 p,u,
substantially lower than the cervical IgG concentration (medi-
IgA SC-lg IgG IgA SC-Ig IgG an, 7.4 ,ug/100 ,ul).
Anamnestic cervical antibody responses after HSV-2 infec-
tion. Subjects 7 and 8 had cervical and salivary IgA and IgG to
HSV at their initial sampling, 3 days after onset of their genital
gc-9 symptoms. Both subjects demonstrated marked increases in
cervical IgA and IgG to HSV-1 and HSV-2 proteins between
L--gE days 3 and 5. New bands were identified in samples from
subject 7 (Table 2) and increased numbers and intensities of
bands were found in samples from subject 8. Further changes
VPl6-, VP16 in cervical IgA or IgG were not observed thereafter. De novo
gD _me - gD IgM to HSV-2 was detected in the cervix as well as the saliva
of both subjects at day 5. IgM then waned in both sites despite

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]*_ [_ AW-
_ICP35 ICP35
FIG. 1. HSV-1-specific cervical and salivary antibody subset pro-
files in a subject with oral HSV-1 infection. Cervical and salivary
specimens from subject 5 were analyzed by Western blotting with
secondary antibodies against human alpha chain (IgA), human secre-
tory component (SC-Ig), and human gamma chain (IgG). Note the
similarities in cervical and salivary IgG profiles and in cervical and
salivary SC-Ig profiles. In contrast, cervical and salivary IgA profiles
differ.
higher than total cervical IgG concentrations (Table 1). Cer-
vical IgA and SC-Ig profiles differed. For example, subject 5
had cervical IgA to gC and to VP16 but lacked IgA to gB and
to gE; her cervical SC-Ig reacted with gB and with gE but not
with gC (Fig. 1). These comparisons suggest that at least two
HSV-specific cervical antibody populations are common, one
with and one without secretory component.
Salivary IgA, IgG, and SC-Ig to oral HSV-1. Salivary IgG,
SC-Ig, and IgA were detected in subjects 1 through 6. As
exemplified by subject 5, IgG-specific salivary and cervical
profiles were virtually identical for each subject (Fig. 1). While
distinct from IgG profiles, salivary and cervical SC-Ig profiles
were virtually identical to one another for each subject (Fig. 1),
as would be predicted by their presumed origin from lympho-
cytes of the common mucosal immune system. In contrast,
differences in salivary and cervical IgA profiles were common.
For example, the cervical IgA profile from subject 5 lacked gB
and ICP35, while the salivary IgA profile lacked VP16 (Fig. 1).
In subjects 1 through 6, the total salivary IgA concentration
was considerably higher than the total cervical IgA concentra-
tion (medians, 52.6 and 14.1 ,ug/100 respectively), while the
p.1,
genital recurrences. In contrast to subjects 1 through 6, sub-
jects 7 and 8 had total cervical IgG concentrations that were
higher than the concentrations of cervical IgA (Table 2),
possibly because of cervicitis and the associated transudation
of serum IgG.
Subjects 7 and 8 developed their full HSV-2 cervical IgA and
IgG profiles coincident with the cessation of HSV shedding on
day 5. Previous studies of seronegative subjects with genital
HSV-2 infections have also demonstrated an inverse correla-
tion between cervical antibodies and the presence of HSV (11).
We recently found that six HSV-seronegative women required
considerably longer (median, 27 days) than subjects 7 and 8 to
develop complete cervical IgA and IgG profiles after genital
HSV-2 infection (2a). These rapid anamnestic cervical anti-
body responses appear similar to those seen with serum
antibodies (1, 2, 9) and in local compartments in animal
models (14). The lesion durations were 11 and 13 days in
subjects 7 and 8, respectively, in contrast to 12 to 38 days in the
seronegative genital HSV-2 patients.
In summary, we detected antibodies to HSV-1 in salivary
and cervical secretions of six HSV-1-seropositive women who
lacked clinical and virological evidence of genital HSV-2
infection. Profiles of local IgA and SC-Ig specific to HSV
differed, suggesting that two HSV-specific antibody popula-
tions develop at mucosal sites, one with and one without a
secretory component. Determining whether these differences
are related to different isotypes or to different forms of IgA
(monomeric and polymeric) or to different epitope-specific
responses requires further studies. We also found evidence for
rapid anamnestic local responses in two additional women with
prior HSV-1 infections. The association between local re-
sponses and resolution of the herpetic episodes in these
subjects suggests that such information may be of value in
identifying the protective mechanisms against mucosal HSV.

TABLE 2. Cervical IgA and IgG specific to HSV-1 and HSV-2 in HSV-1-seropositive subjects with genital HSV-2 infections
Cervical IgA Cervical IgG
Subject Day HSV-1 Total IgA Total IgG
target(s)" HSV-2 target(s)' concnc HSV-1 target(s) HSV-2 target(s) concn'
7 3 None gB NDd gB, gC gB ND
5 All 7 All 7 7.6 All 7 VP5, gB, gC/gE, VP16, 8.4
gD, ICP35
8 3 VP5, gB, gD gB, gC/gE, VP16, gD 2.3 VP5, gB, gC, gE, VP16, gD VP5, gB, gC/gE, VP16, gD 21.0
5 VP5, gB VP5, gB, gG, gC/gE, VP16, gD 13.4 All 7 All 7 44.5
aHSV-1 protein targets were VP5, gB, gC, gE, VP16, gD, and ICP35.
b HSV-2 protein targets were VP5, gB, gG,
gC/gE, VP16, gD, and ICP35.
c Expressed in micrograms per 100 jLl.
d
ND, not done.
5286 NOTES
We thank Julie Dalessio and Patricia Wilson for technical assis-
tance; Michael Remington, Gail Barnum, and Peter Trethewey at the
University of Washington Virology Research Clinic; and Mihee Kim at
the University of Washington Family Medical Center.
This study was supported by NIH grant Al 30731-OlAl.
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