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Division of Infectious Diseases, Children’s Hospital Medical Center, Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH 45229
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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during challenge of HSV-immune mice resulted
in diminished protection of the vaginal mucosa, but not of the sensory
ganglia. These results suggest that the ability to induce vigorous
HSV-specific T cell responses is an important consideration in the
design of vaccines to protect both the vaginal mucosa and sensory
ganglia against HSV-2. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Immunization remains the best hope of preventing HSV disease. Ideally, vaccination would not only protect against disease but prevent infection of the mucosal surface. However, the task of complete protection of the mucosal epithelium against infection seems daunting given that immunization of animals with live attenuated virus or subunit vaccines rarely prevents reinfection with HSV-2 (6, 7, 8, 9, 10, 11). A more realistic goal might be to prevent the establishment of a latent infection of the sensory ganglia and so prevent recurrent HSV disease and asymptomatic virus shedding. In this regard, it has been difficult to demonstrate superinfection of the sensory ganglia with HSV in animal models. Infection of mice (12, 13, 14), rabbits (15), or guinea pigs (8) with attenuated strains of HSV has been reported to prevent or severely reduce the ability of a second HSV strain to latently infect the sensory ganglia. However, the immune mechanisms responsible for this protection have not been identified and understood.
The current study examined the involvement of T lymphocytes in the immune protection of the vaginal mucosae and sensory ganglia of mice previously immunized by intravaginal inoculation with an attenuated HSV-2 strain. The results of these studies suggest that T lymphocytes played an important role in limiting HSV-2 replication in the vaginal mucosa by 24 h after intravaginal reinoculation of HSV-immune mice. Furthermore, T lymphocytes were required for optimal protection of the sensory ganglia of HSV-immune mice, suggesting that HSV-specific T cells, in addition to HSV-specific Ab, are an essential component in the protection of the sensory ganglia of the immune host against HSV reinfection. These results suggest that effective HSV vaccines will need to induce vigorous T cell-mediated immunity in addition to HSV-specific Ab responses to prevent latent infection of the sensory ganglia.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Six- to seven-week-old female BALB/c AnNHsd mice were obtained from Harlan Sprague-Dawley (Indianapolis, IN) and housed in sterile microisolator cages in an American Association for the Accreditation of Laboratory Animal Care (AAALAC) approved facility.
Virus
The bromodeoxyuridine-resistant mutant HSV-2 tk- strain 333, and fully virulent HSV-2 strain 186 were obtained originally from Dr. Lawrence Stanberry (Children’s Hospital Medical Center, Cincinnati, OH) and were grown and titered on Vero cell monolayers, as described previously (7).
Intravaginal inoculation of mice
Mice were inoculated intravaginally with HSV-2 tk- or HSV-2 strain 186 by a modification of the method described previously (16). Briefly, mice were treated twice in a 1-wk period with 3 mg of medroxyprogesterone acetate (The Upjohn Company, Kalamazoo, MI). Mice were anesthetized with sodium pentobarbital, preswabbed, and inoculated using a calcium alginate swab (Spectrum Laboratories, Dallas, TX) soaked with 20 µl of virus suspension. Mice were immunized intravaginally with 5 x 105 plaque-forming units (PFU) HSV-2 tk-. Mice were challenged 4 to 8 wk after the initial HSV-2 tk- inoculation with 5 x 104 to 106 PFU HSV-2 strain 186.
Detection of HSV-specific Ab
Vaginal washes and serum were obtained as described previously (7). For the ELISA assay, samples were plated in duplicate in wells coated with an HSV-2 glycoprotein preparation or a glycoprotein preparation from uninfected cells (mock Ag) as a control for nonspecific binding. Plates were developed using biotinylated anti-mouse IgG Ab (Caltag, San Francisco, CA), peroxidase-conjugated anti-biotin Ab (Vector Laboratories, Burlingame, CA), and o-phenylenediamine dihydrochloride/peroxide (Sigma, St. Louis, MO), as described previously (7). The endpoint titer was defined as the dilution of serum or vaginal wash in HSV-2 glycoprotein-coated wells resulting in an OD490 greater than 0.1 and greater than twice the OD490 obtained from the same sample dilution plated on mock Ag-coated wells.
Virus neutralization assay
Neutralizing serum Ab titers were determined by a modification of the technique described previously (17). Briefly, serum from groups of 6 to 10 HSV-immune or nonimmune mice was pooled and heat inactivated at 56°C for 15 min. A series of twofold dilutions of serum was then made in tubes containing titration media (DMEM + 2% newborn calf serum, 2% penicillin-streptomycin, and 2% amphotericin B) and Low Tox M rabbit C (Accurate Chemical and Scientific, Westbury, NY) at a final dilution of 1/15. Approximately 600 PFU HSV-2 strain 186 was added to each tube containing diluted serum and to control tubes containing titration media plus C (1/15 final dilution) to achieve a final volume of 600 µl. Following incubation at 37°C for 1 h, HSV-2 PFU in each tube was quantified by titration on Vero cell monolayers. The neutralizing titer was expressed as the final serum dilution effecting a greater than 50% reduction in HSV-2 PFU compared with media plus complement alone.
In vivo depletion of immune cells and neutralization of IFN-
NK cells were depleted by i.v. injection of polyclonal rabbit
anti-asialo GM1 Ab (Wako Chemicals USA, Richmond, VA) using the
manufacturer’s recommended dosage, as described previously (16).
Purified stocks of anti-murine CD4 (GK1.5), anti-murine CD8
(2.43), anti-murine Thy-1.2 (30H12), and an isotype control mAb
(SFR8.B6, specific for HLA-Bw6) were prepared from culture supernatants
and used to deplete CD4+, CD8+, or total T
cells by a modification of the method described previously (16).
Depletion of T cells or T cell subsets was verified by flow cytometry
using a Becton Dickinson (San Jose, CA) FACSCalibur analyzer and
CellQuest software at Division of Hematology/Oncology, Children’s
Hospital Medical Center. Briefly, mice were depleted of specific T cell
subsets by daily i.p. injections of 2 mg GK1.5 or 2.43 beginning 6 days
before intravaginal HSV-2 reinoculation. This treatment regimen
routinely resulted in depletion of 80 to 85% CD4+ T and
nearly 100% of CD8+ T cells from the spleens, as measured
on the day of HSV-2 inoculation (data not shown). To deplete total T
cells, mice were injected on alternating days with 1 mg 30H12
(anti-Thy-1.2) or a mixture of Abs GK1.5 and 2.43 (1 mg each). This
treatment resulted in depletion of approximately 90% of
CD4+ and nearly 100% of CD8+ splenic T cells
(data not shown). IFN- was neutralized in vivo, as described
previously (16), by i.p. injection of 2 mg anti-IFN- mAb
(XMG1.2) beginning 1 day before HSV-2 reinoculation and daily
thereafter to the termination of the experiment. Using this treatment
regimen, IFN- was not detected in vaginal secretions of mice on days
1 to 9 following primary HSV-2 vaginal inoculation (16) or on days 1 to
9 in HSV-immune mice rechallenged with HSV-2 (data not shown).
Assay of virus clearance from the vagina and sensory (lumbosacral) ganglia
To quantify HSV-2 present in the vagina, mice were swabbed daily with moist calcium alginate swabs that were stored at -70°C in 1 ml of titration media until titration on Vero cell monolayers, as described previously (7). To quantify HSV-2 present in the lumbosacral ganglia and spinal cords, these tissues were dissected, weighed, and homogenized in 1 ml cold titration media. Cell debris were removed by centrifugation at 5°C, and serial dilutions of homogenates were plated immediately on Vero cell monolayers.
Detection of IFN- in vaginal secretions
Vaginal secretions were collected by washing the vagina twice
with 60-µl vol of HBSS plus 5% FCS. The wash was clarified by
centrifugation and stored at -70°C until assay. The concentration of
IFN- in the vaginal wash was determined by capture ELISA assay, as
described previously (16). Briefly, plates were coated with purified
anti-murine IFN- mAb (R4-6A2) at 5 µg/ml and blocked with PBS
plus 5% BSA. A series of twofold dilutions of rIFN- standards
(Sigma) or undiluted vaginal washes were plated in duplicate and
incubated overnight at 4°C. Plates were washed and incubated with
rabbit anti-murine IFN- Ab (Biosource International, Camarillo,
CA), followed by peroxidase-conjugated goat anti-rabbit IgG Ab
(United States Biochemical, Cleveland, OH). Plates were then washed,
and developed with o-phenylenediamine
dihydrochloride/peroxide (Sigma) in citrate buffer, followed by
OD490 determination. The limit of detection of the assay
was considered to be the last concentration of rIFN- standard, which
gave an OD490 value greater than the mean OD490
plus 3 SDs of wells receiving only diluent.
Statistical analysis
Data were analyzed by one-way analysis of variance with the Bonferroni correction for multiple groups.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Nonimmune mice are exquisitely vulnerable to infection of the
central nervous system following intravaginal inoculation with HSV-2
strain 186. Mice become symptomatic (hunched posture, ruffled fur, hind
limb paralysis) usually beginning on day 5 after inoculation and die of
encephalitis on days 6 to 8 after intravaginal inoculation with HSV-2
strain 186. However, mice survive infection with thymidine
kinase-deficient HSV-2 strains, and the immunity elicited following
primary infection with these HSV-2 strains has been shown to confer
protection against subsequent challenge with normally lethal doses of
wild-type HSV-2 (7, 8, 18). While HSV-specific Ab present in the vagina
may be predicted to protect genital mucosal surfaces by impeding the
initial infection or perhaps enhancing virus clearance, the role of
HSV-specific T cells and innate immune cells such as NK cells in
protection of the vaginal mucosa very early after reexposure to HSV-2
is unclear. To examine the requirement for these cell types in
protection of the vaginal mucosae in HSV-immune mice, NK or T cells
were depleted from mice previously immunized by intravaginal
inoculation with HSV-2 tk- before challenge with the
fully virulent HSV-2 strain 186. As we have shown previously (7),
intravaginal inoculation of HSV-2 tk- resulted in vigorous
HSV-specific Ab responses (Table I
).
HSV-specific Ab titers in serum and vaginal secretions and neutralizing
serum Ab titers were equivalent in T cell-depleted, NK cell-depleted,
and control-treated HSV-immune mice before HSV-2 reinoculation (Table I, Expt. 1). High titers of virus were detected in nonimmune mice
within the first 24 h (Fig. 1), and
these animals failed to clear the virus and usually died by days 6 to 7
after HSV-2 challenge. Despite the presence of HSV-specific Ab,
control-treated HSV-immune mice could be reinfected with HSV-2;
however, virus titers in the vaginae of these animals were reduced
>90% at 24 h after reinoculation compared with nonimmune mice
(p < 0.001), and virus was generally cleared
from the vaginal mucosa by day 4 or 5. Vaginal HSV-2 titers in T
cell-depleted HSV-immune mice were not significantly different from
those of nonimmune animals on days 1 through 6 after HSV-2 challenge.
Identical results were obtained if HSV-immune mice were challenged with
a 20-fold lower dose of HSV-2 (G. Milligan, unpublished results).
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in vaginal
secretions during primary intravaginal infection with HSV-2
tk-, but apparently do not contribute to viral clearance
(16). In contrast, depletion of NK cells from HSV-immune mice before
intravaginal reinoculation with HSV-2 resulted in significantly higher
virus titers on days 1 to 6 compared with control-treated HSV-immune
mice (p < 0.01, p < 0.001,
p < 0.001, p < 0.01,
p < 0.001, and p < 0.05 for days
1–6, respectively), and a delay of greater than 2 days in virus
clearance from the vaginal mucosa (Fig. 1
). Role of CD4+ and CD8+ T cells in protection of the vaginal mucosae of HSV-immune mice
Our previous studies demonstrated that Th1-type
CD4+ T cells predominated the cell-mediated immune
response following primary intravaginal inoculation with HSV-2
tk- and were important for resolution of the primary
vaginal infection, whereas depletion of CD8+ T cells had no
effect on the ability of mice to clear virus from the vaginal mucosa
(16, 19). To determine the importance of these T cell subsets in
protection of the vaginal mucosae of HSV-immune mice against
reinfection with HSV-2, CD4+ and CD8+ T cells
were depleted from HSV-immune mice before virus challenge. As shown in
the previous experiment, HSV-specific vaginal and systemic Ab levels
and neutralizing Ab titers were equivalent among groups before HSV-2
challenge (Table I, Expt. 2). Depletion of CD8+ T cells
from HSV-immune mice resulted in slightly higher mean vaginal HSV-2
titers on each day of infection than were detected in control-treated
HSV-immune mice (Fig. 2). As a
consequence, a slight delay in virus clearance was observed in
CD8--depleted mice. However, depletion of CD4+
T cells from HSV-immune mice resulted in significantly higher virus
titers compared with control-treated HSV-immune mice as early as
24 h after challenge (p < 0.01). Virus
titers remained high in these mice and were not significantly different
from titers of nonimmune mice on days 1, 3, and 4 after reinoculation.
Although CD4+ T cell levels remained reduced through day 8
after inoculation (approximately 91% reduction in CD4+
splenic T cells on day 8 after HSV-2 challenge), HSV-2 titers in
CD4+ T cell-depleted mice fell rapidly after day 4, and
virus was generally cleared from the vagina by days 6 to 7 after HSV-2
challenge.
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in protection of the vaginal mucosae of HSV-immune
mice against HSV-2 reinfection
The elevated vaginal HSV-2 titers in HSV-immune mice depleted of
total T cells (Fig. 1) or CD4+ T cells (Fig. 2)
suggested an important role for T cells in protection of the vaginal
mucosa as early as 24 h after reinoculation. IFN- has been
shown to be essential for resolution of primary cutaneous and ocular
HSV-1 infections (20, 21) and is an important component in conjunction
with CD4+ T cells in the rapid clearance of HSV-2
tk- from the vaginal mucosae of nonimmune mice (16). To
determine when IFN- was present at the site of infection, vaginal
secretions were sampled from nonimmune, T cell-depleted, NK
cell-depleted, and control-treated HSV-immune mice and tested for the
presence of IFN-. As reported previously (16), vaginal IFN- in
nonimmune mice rose 2 days after vaginal HSV-2 inoculation, declined on
day 3, and began increasing again on day 4 (Table II). Vaginal IFN- levels in
control-treated HSV-immune mice were significantly higher than those of
nonimmune mice at 24 h after HSV-2 challenge
(p < 0.001) and continued rising on day 2
before declining on days 3 and 4. Depletion of T cells from HSV-immune
mice before HSV-2 challenge resulted in significantly lower IFN-
production at 24 and 48 h after virus challenge compared with
HSV-immune controls (p < 0.001). NK
cell-depleted HSV-immune mice also had significantly higher IFN-
levels compared with nonimmune mice at 24 h
(p < 0.001) and maintained high levels
relative to control-treated HSV-immune mice on days 3 and 4 after HSV-2
reinoculation.
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in protection of the vaginal mucosa
from HSV-2 reinfection, HSV-immune mice were treated daily with
anti-IFN- Ab beginning the day before HSV-2 challenge. The
results of one such experiment of four performed are shown in Figure 3. HSV-immune mice could be reinfected
with HSV-2, although vaginal virus titers were significantly less than
those of nonimmune mice at 24 h after reinoculation
(p < 0.001). Vaginal HSV-2 titers of
anti-IFN--treated HSV-immune mice varied somewhat among
experiments at 24 h from slightly lower than to slightly higher
than the titers of control-treated HSV-immune mice. However, in all
experiments, HSV-2 titers in anti-IFN--treated HSV-immune mice
rose after the 24-h time point and remained high through at least day 5
after reinoculation.
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Using a guinea pig model of genital HSV-2 infection, Stanberry et
al. (8) showed that a previous genital infection with HSV-2
tk- prevented reinfection of the lumbosacral ganglia
with a fully virulent strain of HSV-2. To extend these findings and
investigate the immune basis for this protection, mice made immune by
intravaginal inoculation with HSV-2 tk- were depleted of T
cells before reinoculation with HSV-2, and the effect on protection of
the lumbosacral ganglia and spinal cord was determined. As shown in
Table III, infectious HSV was detected at
high levels in the lumbosacral ganglia and spinal cord of nonimmune
mice on days 4 to 8 after challenge. In contrast, no infectious virus
was detected during the same time period at either site in
control-treated HSV-immune mice. Depletion of T cells from HSV-immune
mice resulted in the presence of infectious HSV-2 in the lumbosacral
ganglia in two of five mice on day 4, three of five mice on day 6, and
four of four mice on day 8 after reinoculation. Virus titers in
infected ganglia of T cell-depleted HSV-immune mice were slightly lower
than in nonimmune mice on day 4, but were similar on days 6 and 8 after
reinoculation. Similar results were obtained following examination of
spinal cords for infectious HSV-2 (Table III).
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were determined. Based on the kinetics of HSV-2
detection in the sensory ganglia of T cell-depleted HSV-immune mice
(Table III), the presence of infectious HSV-2 in the sensory ganglia
and spinal cords of HSV-immune mice depleted of CD4+ or
CD8+ T cells or treated with anti-IFN- Ab was
determined on days 6 and 8 after HSV-2 challenge. Depletion of
CD8+ T cells from HSV-immune mice before reinoculation did
not abrogate protection of the sensory neurons (Table IV). Similarly, although depletion of
CD4+ T cells or neutralization of IFN- in HSV-immune
mice resulted in high titers of HSV-2 in the vaginal mucosa (Figs. 2 and 3), no infectious virus was detected in either the lumbosacral
ganglia or spinal cord of anti-IFN--treated or CD4-depleted
HSV-immune mice (Table IV).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), it did result in a significant reduction in the magnitude
of HSV-2 replication in the vagina. Importantly, intravaginal HSV-2
tk- immunization resulted in protection of the sensory
ganglia and central nervous system from HSV-2 reinfection, as measured
by the absence of detectable infectious HSV-2 in the lumbosacral
ganglia and spinal cord after intravaginal reinoculation. The results
also demonstrate that HSV-specific T cells played an essential role in
protection within the first 24 h after challenge of HSV-immune
mice. Furthermore, while it is generally accepted that HSV-specific Ab
plays an important role in preventing or limiting the spread of HSV
within the sensory ganglia (24, 25, 26), HSV-specific T cells were also
necessary for optimal protection of the sensory ganglia of
HSV-immune mice.
The involvement of HSV-specific T cells in resolution of HSV infections
has been well documented (25, 27, 28, 29), and as expected, depletion of T
cells from HSV-immune mice abrogated the ability of these animals to
resolve an HSV-2 vaginal infection (Fig. 1). Interestingly, these
results also suggest that the greater than 90% reduction in virus
titer observed in control-treated HSV-immune animals compared with
nonimmune mice within the first 24 h following reinoculation was
not attributable solely to prevention of HSV-2 infection by
neutralizing Ab. Our results suggest that both T cell subsets played a
role in clearance of HSV-2 from the vaginal mucosa. However, the early
T cell-dependent protection was apparently provided primarily by
CD4+ T cells, as selective depletion of this subset from
HSV-immune mice had a more detrimental effect on clearance of virus
from the vagina than did depletion of CD8+ T cells (Fig. 2). Given that mice depleted of both T cell subsets do not clear HSV-2
from the vagina (Fig. 1), we believe the late clearance of virus from
CD4-depleted mice was most likely achieved by infiltrating HSV-specific
CD8+ T cells. Given the low numbers of HSV-specific
CD8+ T cells in this model (19), CD8+ T cells
may not arrive in the vagina in numbers sufficient to affect clearance
until later in the infection. It may be that CD4+ T cells
were responsible for early virus clearance, while CD8+ T
cells were important later to remove remaining foci of infected
cells.
Several mechanisms may account for the role of CD4+ T cells
in protection of the vaginal mucosa. CD4+CTL have been
detected following infection with a number of viruses, including HSV
(19, 30); however, the role of cytotoxicity mediated by these cells in
virus clearance remains uncertain. It is possible that the protection
in the current study may reflect the ability of CD4+CTL
residing in the vaginal mucosa as a result of the initial HSV-2
tk- inoculation to recognize and kill HSV-2-infected
vaginal epithelial cells. In this regard, we have demonstrated
previously CD4+CTL precursors in the genital lymph nodes of
mice following intravaginal inoculation of HSV-2 tk- (19).
Additionally, Parr et al. (31) demonstrated that vaginal epithelial
cells of mice previously inoculated with HSV-2 tk-
expressed high levels of MHC class II Ags 1 day after intravaginal
HSV-2 rechallenge. This increase in MHC class II expression was most
likely the result of exposure to the IFN- present in vaginal
secretions early after HSV-2 challenge (Table II). Although it is
possible that HSV infection may alter MHC class II expression in murine
vaginal epithelial cells, Mikloska et al. (32) have shown that MHC
class II expression by IFN--treated human keratinocytes was not
altered by HSV infection. It therefore seems possible that
CD4+ memory CTL present in the vaginal mucosa as a result
of HSV-2 tk- immunization may have been activated
following HSV-2 reinoculation and recognized processed HSV-2 peptides
in association with MHC class II proteins on infected vaginal
epithelial cells.
While CD4+CTL may play some role in virus clearance, it
seems unlikely that sufficient numbers of effector CTL would be present
over the entire mucosal surface of the vagina within the first 24
h of reinoculation to limit the virus infection. Alternatively, the
protection may be attributable to CD4+ T cell production of
cytokines such as IL-3, TNF-
, or IFN-, which have been shown to
play a role in resolution of HSV infections (16, 20, 21, 33, 34). In
this regard, we detected IFN- in vaginal secretions of HSV-immune
mice 24 h after HSV-2 reinoculation (Table II). The diminished
IFN- levels in T cell-depleted HSV-immune mice compared with NK
cell-depleted mice suggest that the presence of IFN- in vaginal
secretions at 24 h is primarily dependent on T cells (Table II).
We previously showed that neutralization of IFN- delayed resolution
of a primary intravaginal infection with HSV-2 tk- (16).
In the present study, neutralization of IFN- in HSV-immune mice
resulted in a dramatic increase in vaginal virus burden compared with
control-treated HSV-immune mice. Interestingly, the absence of IFN-
during the first 24 h after challenge of HSV-immune mice appeared
to have little effect on vaginal virus titers (Fig. 3). However, as the
infection progressed, neutralization of IFN- resulted in
significantly higher virus titers and a failure to clear the HSV-2
infection by day 6 after intravaginal challenge. These results suggest
that IFN- produced by T cells, although important for the ultimate
resolution of the challenge infection, is not solely
responsible for the T cell-mediated protection observed at
24 h after reinoculation. Other cytokines, such as TNF-, or
granulocyte/macrophage CSF may have acted in synergy with IFN- to
promote protection of the vaginal mucosa by augmenting innate immune
responses or interfering with HSV-2 replication (35).
Delayed-type hypersensitivity mediated by Ag-specific CD4+
T cells has been suggested to play a role in clearance of HSV-1 from
the skin (27). It is possible that this mechanism, induced by
HSV-specific CD4+ T cells, may also be responsible for HSV
clearance from the vaginal mucosae of HSV-immune mice in the present
study. The role of delayed-type reactions in clearance of virus from
the vagina is supported by the infiltration of large numbers of
macrophages and neutrophils into the vaginal tissue soon after
intravaginal challenge of HSV-immune mice (G. Milligan, unpublished
results) as well as the detection of large quantities of IFN- in
vaginal secretions of HSV-immune mice early following intravaginal
rechallenge (Table II). Activation of macrophages and granulocytes by
cytokines such as IFN- or TNF- may result in clearance of HSV by
mechanisms such as direct phagocytosis of virus particles (36, 37),
antibody-dependent cellular cytotoxicity of infected cells (38, 39),
production of nitrogen metabolites (40), or direct lysis of
virus-infected cells (41). It is possible that the diminution of virus
clearance from the mucosa observed following neutralization of IFN-
(Fig. 3) may have been a reflection of insufficient macrophage
activation for efficient viral clearance. The regulation of innate
immune cells such as macrophages and neutrophils by HSV-specific T
cells and their role in protection of the vaginal mucosa are currently
unclear and are the subject of ongoing investigation in our
laboratory.
Depletion of either NK cells (Fig. 1) or CD8+ T cells (Fig. 2) from HSV-immune mice also delayed HSV-2 clearance, but less
dramatically than depletion of CD4+ T cells. These results
suggest that these cells, while capable of effecting virus clearance,
play a lesser role relative to that of CD4+ T cells in
clearance of HSV-2 from the vaginal mucosa of HSV-immune mice. The
contribution of CD8+ T cells and NK cells in HSV-2
clearance from HSV-immune mice contrasts with their apparent lack of
involvement in resolution of a primary HSV-2 intravaginal infection
(16). This difference may reflect greater numbers of these cell
populations residing in the vaginae of mice previously immunized by
intravaginal inoculation of HSV-2 tk- compared with
nonimmune mice. Alternatively, infiltration of these cells into the
vagina may have been enhanced after reinoculation of HSV-immune mice
with HSV-2. NK cell activity in HSV-immune mice may also have been
enhanced by cytokines such as IL-2 and IFN- produced by vaginal
HSV-specific T cells. Because NK cells are present in the vaginal
mucosa before infiltration of HSV-specific T cells following primary
HSV-2 inoculation (26), the lack of involvement of NK cells in
resolution of a primary HSV infection might reflect insufficient T
cell-derived cytokines necessary for complete NK cell effector
function.
Based on the results of studies involving passive transfer of
HSV-specific Ab to HSV-infected nonimmune mice (42, 43, 44, 45, 46) or HSV
infection of B cell-suppressed mice (47), HSV-specific Ab is assumed to
limit the spread of HSV to and within the nervous system. In the
current study, although high titers of HSV-specific, neutralizing Ab
were present in serum and vaginal secretions, T cells were necessary
for optimal protection of the sensory ganglia from HSV infection. While
it is possible that HSV-2 reached the sensory ganglia of
control-treated HSV-immune mice at levels below detection by our
infectious virus assay, the results of these experiments still
demonstrate that effective protection of the ganglia was achieved by
prior intravaginal immunization with an attenuated strain of HSV-2 and
that T cells were responsible in part for this protection. Depletion of
either T cell subset had no apparent detrimental effect on protection
of the sensory ganglia (Table IV), suggesting that the protection could
be provided by either subset. Surprisingly, vaginal HSV-2 titers in
HSV-immune mice depleted of CD4+ T cells or treated with
anti-IFN- were comparable with those of nonimmune mice, although
these mice still displayed effective protection of the sensory ganglia.
These results suggest that multiple immune mechanisms are responsible
for protection of the sensory ganglia and are effective even in the
presence of high titers of virus in the mucosa.
The mechanism by which T cells protect the sensory ganglia is not understood. Passive transfer of HSV-immune lymphocytes has been shown to restrict the acute infection of the sensory ganglia (24) and reduce the number of latently infected neurons (48). Because the protective effect was only observed if transfer of lymphocytes occurred before virus reached the sensory ganglia (24), it is tempting to speculate that T cells function in protection of the sensory ganglia in an indirect manner by effecting rapid viral clearance from the epithelia. Given the ability of HSV to reach the ganglia within 24 to 48 h after inoculation (24, 49, 50), HSV-specific memory T cells must be activated and provide their effector function very soon after virus inoculation to provide significant protection of the ganglia. The results of the T cell-depletion experiments in the current study are consistent with this assumption and suggest that vaginal T cells primed by an initial intravaginal HSV infection play a critical role in virus clearance from the genital mucosa by the first 24 h of reinoculation. HSV-2 titers in the vaginae of control-treated HSV-immune mice were reduced greater than 90% compared with nonimmune or T cell-depleted HSV-immune mice at 24 h after reinoculation. At these lower virus titers, neutralizing Ab or innate immune mechanisms may be sufficient to protect the nerve endings from HSV-2 infection.
It is also possible that HSV-specific T cells act within the sensory ganglia to clear virus that escapes neutralization by HSV-specific Ab. T lymphocytes have been shown to infiltrate sensory ganglia by 5 days after primary HSV inoculation and remain at this site for at least 6 mo (51, 52). Furthermore, CD8+ T cells that infiltrate the ganglia following cutaneous HSV-1 infections play a role in virus clearance from the peripheral nervous system by a nonlytic mechanism (53). In the present study, T cells that infiltrated the sensory ganglia as a result of HSV-2 tk- immunization may have prevented superinfection of the ganglia by the challenge virus in a similar fashion. Experiments are currently underway to further establish the mechanisms by which T cells cooperate in the protection of the sensory ganglia as well as to identify the anatomical sites at which they act.
Mucosal Ab may serve as a barrier that prevents or limits the infection of mucosal cells. The present studies did not directly address the role of neutralizing Ab in protection, but do demonstrate effective protection of the vaginal mucosa and sensory ganglia in the presence of both HSV-specific T cells and Ab. These results suggest that HSV-specific T cells in the vaginal mucosa were required to clear virus that penetrated the mucosal Ab barrier. In fact, the data are consistent with a model in which HSV-specific T cells and Ab work synergistically to limit the infection and clear virus that penetrates the Ab barrier in vaginal secretions. In this regard, passive transfer of an HSV glycoprotein D-specific mAb has been shown to prevent HSV-1-induced encephalitis in the absence of one, but not both, T cell subsets (54).
The very rapid T cell response to HSV-2 intravaginal reinoculation in these studies may reflect the presence of virus-specific memory T cells in the vaginal mucosa. In this regard, we have shown previously that HSV-specific memory T cells reside in the vaginal mucosa for at least 2 mo following intravaginal HSV-2 tk- inoculation (19). The results of the current studies indicate that very efficient protection of the vaginal mucosa and sensory ganglia against HSV-2 infection may be achieved by intravaginal immunization. Because the vagina is a poor immune inductive site for nonreplicating vaccines (55), further studies are currently underway to determine whether similar levels of humoral and cell-mediated immune protection can be provided to the vaginal mucosa and sensory ganglia by immunization at alternative sites.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Gregg N. Milligan, Division of Infectious Diseases, Children’s Hospital Research Foundation, 3333 Burnet Avenue, Cincinnati, OH 45229.
3 Abbreviations used in this paper: HSV, herpes simplex virus; PFU, plaque-forming unit; tk, thymidine kinase.
Received for publication November 20, 1997. Accepted for publication February 17, 1998.
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enhances resolution of herpes simplex virus type 2 infection of the murine genital tract. Virology 229:259.[Medline]
(IFN-). Virology 202:76.[Medline]
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against experimental infection with herpes simplex virus type 1. J. Gen. Virol. 72:143.. Virology 180:822.[Medline]
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